A Study Of Tissue Engineering Bone-Based On Cell-sheet Techniques

Tissue engineering bone provides a new method for the rehabilitation of bone defects which are caused by tumor resection,trauma and inborn deformity. How to find the cell seeds is the key problem of the bone tissue engineering.We chose bone marrow stromal osteoblast cells as cell seeds in this study.These cells were cultured in vitro to construct bone marrow stromal osteoblast cell sheet.A new method for constructing polimer of bone marrow stromal osteoblast cell----Bone marrow stromal osteoblast cell sheet was proposed.The main techniques of this method are:①extracting stem cells of bone marrow stromal cells;②amplifying stem cells of bone marrow stromal cells;③stem cells of bone marrow stromal cells are constructed into bone marrow stromal osteoblast cell sheet;④bone marrow stromal osteoblast cells are growing in three dimensiosn on the surface of matrix secreting from itself.The feature of this method is:①the constructed bone marrow stromal osteoblast cell sheet is divided into three layers,substantia propria layer,and stratum intermedium and cellular layer.②The multiplied cells are keeping good cell type and has the ability of differentiation.③good biological compatibility and low immunogenicity(immunogenicity comes from fetus bovine serum).④The high utilization efficiency of cell seeds.⑤Certain shape tissue could be constructed by the help of the scaffolds of natural materials.In this study,the physico-chemical property,histological construction and biochemical ingredients of bone marrow stromal osteoblast cell sheet were analyzed.The ability of forming neo-bone in vitro and forming certain shape bone in vivo of bone marrow stromal osteoblast cell sheet were evaluated by three dimensiosn reconstructing in vitro and animal experiment.Furthermore,a new method of constructing bone marrow stromal osteoblast cell sheet is proposed in order to providing a better way for constructing transplantation in vitro to rehabilitating bone defect.Experiment 1:the in vitro construction of bone marrow stromal osteoblast cell sheetObjectives:In this study,the histological construction and biochemical ingredients of bone marrow stromal osteoblast cell sheet were analyzed.The ability of forming neo-bone in vitro and forming certain shape bone in vivo of bone marrow stromal osteoblast cell sheet were evaluated by three dimensions reconstructing in vitro and animal experiment.Methods:the in vitro construction of bone marrow stromal osteoblast cell sheet:Leporine BMSCs were aspirated from the bone marrow,gradient centrifuged and plated into six petri dishs(nine centimeter in diameter).At 80%confluence,culture media was changed to osteogenic induction media at.At 100%confluence, digesting and recollecting cells,then plate these high density of cells into 3 petri dishs(nine centimeter in diameter).These cells were cultured in petri dish for 14 days to detecting the forming of bone marrow stromal osteoblast cell sheet.At the same time,tissue staining and electron microscope were used in observing cell sheet.Results:after in vitro culturing,bone marrow stromal osteoblast cell sheet is a semi-transparent,elastic membrane,rich in hard white nodule and can be clipped by nippers.H.E.staining confirmed neo-bone and bone lacuna like tissue forming.Von kossa staining confirmed neo-calcified tissue forming in matrix.The bone marrow stromal osteoblast cell sheet is divided into three layers,substantia propria layer,stratum intermedium and cellular layer by electron microscope scanning.Conclusions:two polar hypothesises(quiescence polar and proliferation polar) of bone marrow stromal osteoblast cell sheet is proposed.A new technique of constructing bone marrow stromal osteoblast cell sheet is established.Bone marrow stromal osteoblast cell sheet is a combination of osteoblast of bone marrow stromal cell and matrix(self secreting).It is believed that the bone marrow stromal osteoblast cell sheet will be valuable in autogenous bone transplantation in vitro.Experiment 2:neo-bone formation in the subcutaneous position of nude rat with coral combined with bone marrow stromal osteoblast cell sheetObjectives:The ability of bone marrow stromal osteoblast cell sheet developing into tabulate bone like tissue in aphylactic animals was detecting under the new technique of constructing bone marrow stromal osteoblast cell sheet.Methods:After cultured in osteogenic medium for 2 weeks,the sheets were then wrapped around the pre-seeded scaffolds made of tabulate coral.The quiescence polar of the bone marrow stromal osteoblast cell sheet was toward to natural tabulate coral.The fusion of the cell sheet with tabulate coral was detecting after 4 weeks culturing in vitro.The maturity of the new constructing complex was detecting by histological staining.Then the constructs consisting of cell-sheets assembled with tubulate coral were transplanted subcutaneously into nude rats.The samples were divided into 8-week group and 12-week group in order to detecting the ability of bone formation.Results:After 4 weeks culturing in vitro,there was mature forming and tight fusion between the outer of the coral and the cell sheet.And there was mature bone forming in the middle of the tabulate coral.Conclusions:Bone marrow stromal osteoblast cell sheet has the ability of developing into mature bone under good trophallaxis circumstance.Adjusting the face of the wrapping cell sheet(the face of the quiescence polar and proliferation polar) is providing a new method for constructing three dimensionss of the bone marrow stromal osteoblast cell sheet.Experiment 3:neo-bone formation in the subcutaneous position of nude rat with coral transplantation combined with bone marrow stromal osteoblast cell sheetObjectives:The new transplantation containing bone like tissue was constructed under the technique of bone marrow stromal osteoblast cell sheet in order to explore the feasibility of constructing transplantation containing bone like tissue in vivo.Methods:Bone marrow stromal osteoblast cell sheet was constructed following the procedure of experimetnt 1.the sheets were then wrapped around the pre-seeded scaffolds made of tabulate coral.The proliferation polar of the bone marrow stromal osteoblast cell sheet was toward to natural tabulate coral/transplantation.The fusion of the cell sheet with tabulate coral was detecting after 4 weeks culturing in vitro.The maturity of the new constructing complex was detecting by histological staining.Then the constructs consisting of cell-sheets assembled with tubulate coral/transplantation were transplanted subcutaneously into nude rats.The samples were divided into 8-week group and 12-week group in order to detecting the ability of bone formation.Then the transolantation was taken out of the body to evaluate the bone formation from general shape,histology construction etc.Results:The bone bone marrow stromal osteoblast with coral/transplantation has good biological compatibility.The previous shape of the complex is unchanged,has similar construction of natural bone and abundant calcified minerals and collagen fibers.Conclusions:Bone marrow stromal osteoblast cell sheet has good ability of forming well bone like tissue in vivo.The construction and cell type of the neo-bone is like the natural bone.So it provides a good solution for rehabilitating of bone defects.Experiment 4:The study of the bone formation ability of transplantation constructed of the bone marrow stromal osteoblast cell sheet sticking to the scaffoldsObjectives:In order to find the techiniques of bone formation of bone marrow stromal osteoblast cell sheet sticking to the scaffolds.Methods: Bone marrow stromal osteoblast cell sheet was constructed following the procedure of experimetnt 1.Then the cell sheet was put on one side of the round plate coral.After short time culturing in vitro,there is a tight junction between the cell sheet and the round plate coral.Then the newly constructed complex was transplanted into the face of the skull of Newzealand rabbit without periosteum.The specimens were taken out of the body after 8 weeks and 12 weeks in vivo culturing of different group.The ability of neo-bone formation was detecting in general shape,histology construction etc.Results:A lot of neo-bone with certain mechanical function was obtained from the above methods.Conclusions:The neo-bone forming from this technique is different from other techniques.The neo-bone forming from this technique is from bone marrow stromal osteoblast cell sheet and without bone resorption.But the neo-bone formation from other technique has certain bone resorption.Experiment 5:The study of the use of the bone marrow stromal osteoblast cell sheet in rehabilitating maximum skull defectObjectives:The use of the bone marrow stromal osteoblast cell sheet in rehabilitating certain shape defect(the maximum skull defect of rabbit) of bone tissue under no supporting materials.Methods:Bone marrow stromal osteoblast cell sheet was constructed following the procedure of experimetnt. The cell sheet was transplanted into the maximum skull defect of rabbit.At the same time,we chose auto-ilium transplantation as positive controlled group,and the coral transplantation as negative controlled group.After 8 weeks and 12 weeks in vivo,the ability of rehabilitation of the 3 group was evaluated by X ray examination,HE staining and Masson's three color staining.Results: Histological examing shows that the neo-bone forming from bone marrow stromal osteoblast cell sheet has good quality and is fit for physiological requirements.The ability of rehabilitating maximum skull defect of the cell sheet is as good as auto-ilium transplantation.The coral transplantation has no ability of forming new bone.Conclusions:The tissue engineering bone made from bone marrow stromal osteoblast cell sheet is a promising material for rehabilitating bone defect.Experiment 6:the ability the transplantation combined the bene marrow stromal osteoblast cell sheet and the coral in rehabilitating mandible defect of the goatObjectives:The mass bone with certain shape was constructed under the new technique of bone marrow stromal osteoblast cell sheet in order to rehabilitating mandible defects in large animals.Methods:We chose goat as our experiment animal.Bone marrow stromal osteoblast cell sheet was constructed following the procedure of experimetnt 1.The coral was provided as a mandible like scaffold material to construct a new transplantation.There are implantations in the coral.The cell sheet was supported by the scaffold.The proliferation polar of the cell sheet was faced to the scaffold.After 4 weeks cultured in vitro,the new transplantation was transplanted into the mandible defect of the experiment animal.The rehabilitation of the bone defect in animal wan evaluated by general shape,histological examination after the new transplantation was put in the defect position for 12 weeks.Results:Through this method,the transplantation has stable shape to conform to the defect of the animals.Histological examination shows the good quality of the neo-bone,and the neo-bone is fit for the requirements of the physiological need.Conclusions:The mass bone with certain shape constructed under the new technique of bone marrow stromal osteoblast cell sheet with inner supporting is promising in rehabilitating large bone defects.