| Introduction:On account of implant denture possessing some outstanding advantages instance of artistic, high efficiency and no harm to adjacent teeth, it has been increasingly accepted by both doctors and patients. As a treatment option, immediate implant placement following tooth extraction has been recommended, particularly for the replacement of anterior teethfor several advantages such as optimal positioning of the implant, reduction of post extraction resorption and shortening the treatment time. However, the discrepancy between the diameter of the socket and the implant usually generates a gap between the bony walls of the socket and the implant, where is the widest in the coronal part of the recipient site. Most studies indicated that marginal defects around implants (with all bony walls intact) less than2mm were thought to be negligible and had no need for bone augmentation.In fact, most dentists often handle with large bony defect peri-implant which is more than2mm in clinical.In large bony defects, epithelial cells can be prior to colonizing the gap, which induces fibrointegration but not osseointergration and results in implant failure at last.In order to prevent fibrointegration and achieve implant osseointegration at immediate implant, numerous investigations have been conducted. Guided bone regeneration (GBR) has become an important therapeutic procedure for bone and peri-implant defects, as well as bone augmentation procedures prior to implant placement. GBR alone or in association with bone replacement graft had been recommended in gaps wider than2mm. However, it has disadvantages like needing a second removal operation of non-resorbable membranes, being unable to produce sufficient bone formation with resorbable membranes such as collagen membrane.The development of bone tissue engineering offers a hopeful opportunity for peri-implant bone defect repair. Three basic biological factors involved in bone tissue regeneration which are seed cells, differentiation inducing factor and scaffolding materials. Used as seed cells, mesenchymal stem cells come from bone marrow (BMSCs) has been used in the past years. But the drawbacks of its in collecting cells, aging, high viral pollution and limited proliferative property restrict the utility in bone tissue engineering.As new and potential seed cells, GMSCs have been paid extensive attention in tissue engineering. Besides its ease of isolation and no viral infection, GMSCs are of higher proliferative capacity compared with BMSCs and have no drawbacks of embryonic stem cells such as source deficiency, xenoma rejection and ethical concerns.PRF, a second generation platelet concentrate, can be manufactured in a simple procedure. As containing varied growth factors such as platelet-derived growth factor (PDGF), fibroblast growth factors (FGFs), epidermal growth factor (EGF), transforming growth factor-beta (TGF-b) and so on. PRF has been widely used in bone tissue engineering. Animal experiments and clinical trials have proven that PRF has great potential in promoting the generation of the gingiva, alveolar bone and cementum in a variety of wound healing models. Our previous work also showed PRF could promote the osseointegration of the immediate implants.To our knowledge, there have been no studies investigating the effect of GMSCs on repairing peri-implant bone defect post immediate implant.In this study, we created a bone defect adjacent to the implant after immediate implant. GMSCs were injected into the defect. Then the peri-implant defects were filled with PRF in beagle dogs. The purpose of the present study was to evaluate the influence of GMSCs on bone defect regeneration peri-implant after immediate implant.Materials and methods:1. Isolation and cultivation and differerntiation of GMSCs GMSCs at passage4were incubated with antibodies against human CD34, CD45, CD105, CD29and CD90for flow cytometry analysis. CFU-F assay was performed to assess the colony-forming efficiency of GMSCs. To assess the multipotent differentiation potential, GMSCs were incubated in a-MEM containing5%fetal calf serum (FCS),10mM β-glycerophosphate,50mg/L ascorbate-2-phosphate and0.1μM dexamethasone for osteogenic differentiation induction;10%FCS,1μM dexamethasone,200μM indomethacin,10μM insulin and0.5mM isobutyl-methylxanthine (IBMX) for adipogenic differentiation induction and10%FCS,50nM ascorbate-2-phosphate,0.1μM dexamethasone for chondrogenic differentiation induction. Alizarin red staining, oil red O staining and toluidine blue staining were performed to examine the osteogenic, adipogenic and chondrogenic differentiation respectively.2. Imimediate implant and creat bone defect peri-implantAfter extracting the second, third and fourth premolars bilateral mandible, dental implants were inserted into the distal root extraction socket immediately and the torque was35N.cm. Special care was taken to avoid injury to the bony wall and the dental implants. On the basis of implant surgical principles, bone defects peri-implant were created adjacent to the implant with4mm in height,4mm in the mesio-distal direction and3.5mm in the bucco-lingual direction after immediate implant. All dental implants gained the primal stability. In the experiment group, the4th passage UCMSCs (Lifeline Cell Technology, FC-0020) re-suspended in fresh a-MEM at1×10/ml, were injected into the marrow cavity of the defect area and filled with PRF. The peri-implant defects in the control group were filled with PRF only.Results:1. Isolation and cultivation and differentiation of GMSCsFlow cytometry analysis showed that the4th generation GMSCs expressed CD105+(85.14%), CD29+(95.11%), CD90+(91.78%),Stro-1+(16.21%),CD34-(0.09%) and CD45’(0.03%),which are markers for MSCs, indicating the cultured GMSCs were of high purity. CFU-F assay showed GMSCs had strong colony forming ability. After osteogenic, adipogenic and chondrogenic differentiation induction, mineralized nodules, accumulated lipids and the positive staining density were detected with alizarin red staining, red O staining and toluidine blue staining respectively, which indicated that GMSCs owned the multipotent differentiation potential.2. Imimediate implant and creat bone defect peri-implantOsteoblast proliferation was detected in the test group at the early stage of new bone formation. By the second week, almost no new bone tissue was visible on the surface of dental implant in the defect area in both groups. However, some newly bone formation could be detected at the bottom of the defect area and near the host bone. The BIC in the2nd week showed experiment group (19.38±1.17%) was similar to control group(17.51±2.15%) and there was no significant difference between groups. The percentage of newly formed bone wasl7.08±0.49%in the experiment group and14.30±1.25%in the control group, manifesting a significant difference between groups.In the4th week, the quantity of newly formed bone in contact with dental implant surface was increased at this period in both groups. Most of the bone-implant interfaces contact directly, mass of newly formed bone occupied the defect region in experiment group. In control group, reticular formation by new formed trabecular structures occupied the defect area. BIC measurements in the experiment group was51.08±0.98%and the control group was40.79±0.65%, indicating a significant difference between groups. The percentage of newly formed bone in the experiment group was47.33±3.21%and the control group was37.04±2.29%, indicating a significant difference as well. In the8th week, bone tissue was more mature than before. In experiment group, bone to implant interface was closely contacted, and Haversin system could be identified. BIC measurements was72.83±1.09%in the experiment group and61.17±2.79%in the control group, showing a significant difference between groups. The percentage of newly formed bone was65.96±3.6%in the experiment group and58.83±3.36%in the control group, showing significant difference as well. Conclusions:1. GMSCs have the similar characteristics with other stem cells, including possess strong colony forming ability and multipotent differentiation potential.2. GMSCs can repair bony defect peri-implant after immediate implant and can be ideal candidate of seed cells for bone regeneration. |
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