| ObjectiveCongenital aplasia, maxillofacial trauma, tumor, surgery that lead to the large bone defects seriously affect the people’s survival and quality of life. Large block of maxillofacial bone defect repair has been a difficulty of maxillofacial surgeon clinically. The use of traditional bone defect repair method (including autologous or allogeneic bone graft, synthetic materials, etc.) to achieve a considerable therapeutic effect, but there are still many shortcomings unavoidable. Distraction osteogenesis(DO) is one of an endogenous bone tissue engineering new technologies to generate new bone tissue of which mechanism is the utilization of the bone callus after trauma healing,and the technology in craniofacial surgery, cancer reconstruction surgery, dental slot planting areas show the broad application prospects. However, TDO that must make a suitable bone transport disc near the bone defect area can be carried out with a treatment, and it will be ineffective for the patients with severe bone defects or severe bone deformity and bone defects can’t be fitted with a suitable bone transport disc. How to solve this problem, and let distraction osteogenesis have a broader application prospects, which has been a hot research for many scholars. Zhou Nuo(professor and instructor) using free autogenous bone transport disk (ie, fully periosteal stripping, no soft tissue attachment) for distraction osteogenesis successfully build the animal model of "non-vascularized transport disc distraction osteogenesis of mandible", and internationally firstly proposed the new model of "non-vascularized transport disc distraction osteogenesis", which provides a new way of thinking for large bone defects repair that can’t treated through producing proper bone transport disc with its own bone in bone deficit area. However, autologous free bone transport disc still need donor bone in donor site, and if a new bone transport disc instead of autologous bone as free transport disc, avoiding secondary trauma of the donor site and reducing the pain of patients caused by surgery.Therefore, on the basis of animal model of "non-vascularized transport disc distraction osteogenesis", this study will use cell sheet technology, with combination of seed cell engineering, genetic engineering and bone tissue engineering, to make bone marrow mesenchymal stem cells (BMSCs) which stablely express bone morphogenetic protein-2(hBMP-2) into cell sheets, and then will be constructed with freeze-dried bone to produce free tissue engineering biological transport disc, which will be finally used to undergo transporting disc distraction osteogenesis repair in mandibular bone defect area. Generally, this study further explore a new mode of distraction osteogenesis feasibility through building a new kind of biological transport disc, and to explore the mechanism of bone formation. Simultaneously, naturally occurring process of embryonic bone formation is simulated, through the pathway that cell sheets controllably sustained expression of BMP-2at the desired time, and pluripotent stem cells and fibroblasts in transport disc or ends zone of distraction osteogenesis own bone will be induced into bone or cartilage cell differentiation, so that osteogenic cascade will start again and renew directed osteogenic differentiation, and ultimately to promote new bone formation and remodeling in bone defect area, thus open another new model of bone distraction, and provide a theoretical basis for further widely used in the clinical treatment of distraction osteogenesis for a variety of severe bone defects or bone deformities.Methods1The experimental study of canine bone marrow mesenchymal stem cells (BMSCs) separation, cultivation and identification in vitro:BMSCs were aspirated from baby dog tibia’s bone marrow and isolated by the whole bone marrow adherence method, and identified by cell morphology and cell surface antigen. Then they were induced their differentiation into osteoblasts and chondroblast which were identified by histochemistry staining and Trace enzyme standard method.2The experimental study of integrating hBMP-2into BMSCs by lentivirus gene transfection:Take passage2BMSCs, with lentivirus as gene avector for integrating BMP-2gene into BMSCs, and transfection rate was by detected by Flow cytometry instrument, and the cell proliferation and growth of the ability after transfection were tested, and the expression of hBMP-2mRNA was detected by RT-PCR. G418was used to sift the stable expression cells for two weeks after transfection. And then the expression of BMP-2protien was tested by western blot and immunochemistry.3BMSCs cell sheet’s preparation and its biological characteristics research: Taken passage2BMSCs, the cells were cultured in a6cm culture dish with the density of1×106till7～10days after the formation of cell sheet. The biological characteristics of cell sheet was observed through the inverted microscope, HE staining, immunochemistry, TEM ect. Dog BMSCs cell sheet and enzyme digestion of dog BMSCs were compared with the expression level of collagen I and fibronectin in the extracellular matrix by real-time fluorescent quantitative polymerase chain reaction (qPCR).4Preparation of freeaze-dried boneand evaluation of the cellular compatibility: Canine freeze-dried bone was made through block preparation, deep frozen degreasing, freeze drying and sterilization. BMSCs were cultured on FDB in vitro. The tissue structure of FDB was observed by HE staining. The influence of FDB on the proliferation of BMCSs was observed by MTT method. The adhesion and growth of BMSCs were observed by scanning electron microscope (SEM).5hBMP-2modified BMSCs cell sheet composite freeze-dried bone as a biological transport disc in canine mandibular TDO experiment research: Eighteen healthy mongel dogs were randomly divided into3groups, and6dogs of each group. A-group is experimental group for hBMP-2modified BMSCs cell sheet composite freeze-dried bone as a biological transport disc. B-group is for BMSCs cell sheet composite freeze-dried bone as a biological transport disc. And C-group is for freeze-dried bone as a biological transport disc. A animal model of TDO for canine mandible was constructed. Each experimental group will fix the biological transport disc that had prepared before the operation of TDO. On the time point of two weeks, four weeks and eight weeks after distracting in TDO, the formation of new bone and reconstruction of bone was observed by general pathology, morphology, and X-ray test, and the expression of BMP-2protein was detected by immunochemistry. Results1The results of the experimental study of canine bone marrow mesenchymal stem cells (BMSCs) separation, cultivation and identification in vitro:The primary and passages of BMSCs spindle in shape had adhesive character, and grew in colonies. These cells were positive for essential MSC surface molecules (CD90, CD44, CD29) and negative for most haematopoietic markers (CD34, HLA-DR). The result of alizarin red staining was positive after differentiation to osteogenesis. In addition, the result of HE staining show cartilage tissue after differentiation along chondrogenic pathways. And the result of toluidine blue staining showed that cartilage extracellular matrix was dyed indigo blue after differentiation to chondrogenesis.2The results of the experimental study of integrating hBMP-2into BMSCs by lentivirus gene transfection:Through use lentivirus as gene avector for integrating BMP-2gene into BMSCs, the transfection rate is about70percent of cells, and the green fluorescence was seen after48h transfection, and the intensity of the green fluorescence was strongest after96h transfection. The results of MTT showed the growth proliferation capacity of the canine BMSCs after transfection was no change. The results of RT-PCR show the expression of hBMP-2mRNA in the canine BMSCs after transfection. G418has successfully shift the stable expression the canine BMSCs. The expression of BMP-2of these cells could be detected after4weeks culture by immunochemistry and western blot.3The results of BMSCs cell sheet’s preparation and its biological characteristics research:Under the invert microscope we can see that the cell of BMSCs cell sheet group as multilayer, the cell are long fusiform and full fusion. The cell boundaries were not clear. By HE staining we can look the BMSCs cell sheet made of multilayer cells, and compact connection between cells. By SEM we can observe compact connection between cells, the cell group and differentiation are very good. We can see the extracellular matrix and collagen fiber around the cells. The results of qPCR show canine BMSCs cell sheet are more expression of COLL I and fibronectin(p＜0.05). The cell sheet is detected the expression of COLL I and fibronectin by immunochemistry.4The results of preparation of freeaze-dried bone(FDB) and to evaluate the cellular compatibility:The freeze-dried bone has been successfully prepared. The results of MTT showed that canine FDB had no influence on the proliferation of BMSCs. Under the SEM, the BMSCs adhered on the surface of the canine FDB tightly, and they grew well and showed polygon shape.5The results of hBMP-2modified BMSCs cell sheet composite freeze-dried bone as a biological transport disc in canine mandibular TDO experiment research:All experimental animals were successfully completed with a surgery of transport distraction osteogenesis. Operation area and the mouth without a passageway. The operation incision had no infection. The animal model can successfully be distraction. The results of general pathology, tissue section HE staining and X-ray examination show that the quantity of regeneration of new bone ranked as follows:group A> group B> group C. The gray value of the results of IHC showed that the expression of BMP-2of group A was obviously higher than that of group B and group C (p<0.05), and group B was higher than group C (p<0.05).Conclusions1hBMP-2modified BMSCs cell sheet of freeze-dried bone allograft has been successfully built as a new biological transport disc. 2The feasibility of this TDO model that used "hBMP-2modified BMSCs cell sheet of freeze-dried bone allograft as a new biological transport disc" has been Verified.3hBMP-2modified BMSCs cell sheet can promote the formation of new bone. |
PDF Full Text Request