Osteoblastic Differentiation Of Rabbit Adipose-derived Stem Cells Transfected By Polyethylenimine-mediated Bone Morphogenetic Protein-2Gene In Vitro

Bone defect was common in clinical which caused by trauma, infection, tumor, congenital diseases and so on. Due to the limitation in bone mass and complications of the donor site, autogenous bone transplantation couldn’t meet the needs of the treatment of bone defect, which was the gold standard in the treatment of bone defect. The rising of tissue engineering brought new hope for treatment of bone defect. Bone tissue engineering was an ideal treatment method by new bone tissue to repair bone defect, using seed cells with osteogenetic ability or potential implanted in the body after compound biomaterials in vitro. Tissue engineering had three elements, namely the seed cells, inducing factors and carrier. The seed cell was one of the most basic and primary link. Stem cells were the most ideal tissue engineering seed cells because it had a potential to self-replicate and multidirectional differentiate. At present, main sources of stem cells were from skeletal muscle satellite cells, embryonic stem cells, bone marrow-derived mesenchymal stem cells and so on. Skeletal muscle satellite cell was low content in muscles, in addition, muscles were hard to harvest. Source of embryonic stem cells had immune rejection and ethical issues. Bone marrow mesenchymal stem cells had been accepted by most scholars as seed cells in bone tissue engineering, past researches about bone tissue engineering seed cells more concentrated in bone marrow mesenchymal stem cells, but its shortcomings were not easy to accept by patients,such as less quantity, hard harvesting, difficulty in vitro cultivation and easy ageing, and so on.Adipose-derived stem cells(ADSCs) had became a hot spot after bone marrow stem cells since Zuk isolated a stem cells with multidirectional differentiation potential extracted from adipose tissue suspension in human liposuction for the first time in2001. Studies showed that ADSCs could differentiate to the bone, cartilage, fat, muscle, cardiac muscle cells, nerve cells, epithelial cells and liver cells under specific induction condition in vitro. ADSCs might be as an ideal seed cells to construct tissue engineered bone with many advantages such as autologous sources, easy harvest, adequate cell contents, multidirectional differentiation, genetic stability, autologous transplantation without immune rejection and without medical ethics problem.Inducing factor was one of the three elements in tissue engineering, the source of the inducing factor of bone tissue engineering were mainly three ways:directly join the inducing factor, slow-release inducing factors through carrier and release inducing factor by target cells themselves expressing using gene therapy techniques, etc. The first two were both the exogenous factor by simple administration, but formal studies of tissue engineering showed that using cell factor existed disadvantages such as complex procedure, low yield, unstable activity both in natural biological purification and in genetic engineering expression, so the difficulty and hotspot in the research of the tissue engineering had always been how to effectively play the utility of inducing factor timely and moderately, and to give play to the normal physiological function to become the ultimate ideal substitute through specific absorption between cells and substrate, cell proliferation, directional differentiation. the combination of genetic engineering and tissue engineering provided a good method to select for the solution of the above problems. In order to meet the needs of directional differentiation of seed cells, gene technology in the application of tissue engineering was mainly to transfect target inducing factor gene into seed cells for sustained releasing of inducing factor. Inducing factor was the deciding factor of directional differentiation of stem cells. Bone morphogenetic protein (BMP) was members of the super family of transforming factor beta, at present, which had been found more than20subtypes in total. BMP-2was the specific factor of bone growth factors, which had been known to induce stem cells to osteogenetic differentiate alone, and widely regard as one of strongest osteogenetic inducing factor. Their gene recombination product rBMP-2was the only osteogenetic inducing factor approved by the American Food and Drug Administration to be used in clinical. But the disadvantages of exogenous factor rBMP-2greatly influenceed the effect of osteogenesis and limited its wide application in clinic, such as easy to spread, quick degradation, short half-life, large dosage, expensive etc. BMP-2gene Modification of seed cell mad cells to express inducing factor endogenously by gene transfection technology, which could overcome the above shortcomings, and solve one of the challenges in bone tissue engineering--induceing factor problem.One of the key problems involved in the application of gene transfection technology was how to choose appropriate gene transfection carriers. At present the commonly used gene carriers included two kinds:viral carrier and non viral carrier. The viral carrier was mainly used in the current researches and clinical trials. But viral carrier had some defects difficult to overcome, such as the complex preparation, poor targeted specificity, high immunogenicity, unallowed repeated use in vivo, potential risks of causing infection and cell malignant transformation and so on, which limited the application of viral carrier in clinical. Therefore, more and more researchers had put their eyes on non viral carriers in recent years. Non viral carrier had many advantages, such as simple preparation, low cost, easy quantity production, large capacity of carrying genetic material, good security, but also had some shortcomings, such as the low transgenic efficiency, in and of itself, without specific cells or tissues targeting. Polyethylenimine (PEI) was a new type of gene carrier with cationic polymer, as a kind of non viral carrier with high transgenic efficiency, it had become a "gold standard" of non viral carrier to measure efficiency of other kinds of carriers in the area. We used PEI and ADSCs as gene transfection carriers and seed cells respectively in this paper, and wanted to provide theoretical basis and experimental evidence for the treatment of bone defects and nonunion, through transfecting inducing factors BMP-2gene into target cells ADSCs, making cells sustained release inducing factor BMP-2, and then inducing seed cells to osteoblast differentiation, further constructing tissue engineering bone.This paper was divided into three parts. The first part was the experimental research of the basic biological characteristics and multidirectional differentiation of the rabbit adipose-derived stem cells (ADSCs). We cultivated ADSCs in vitro and amplified by passage in order to explore the ideal cultivating method of rabbit ADSCs in vitro. We Observed the cell morphology and growth characteristics of ADSCs of the original and passage generations in order to explore the biological characteristics of ADSCs. We identified the phenotypic characteristics of ADSCs and measured the influence of passage culture in vitro and osteogenetic differenciation induction on the phenotype of ADSCs, in order to explore the influence of passage culture on proliferation, differentiation and apoptosis of ADSCs in vitro. We also induced ADSCs of passage3and6generations into fat and bone differentiation in order to evaluate the multidirectional differentiation potential of ADSCs, further confirm that the cells had the properties of stem cells, and explore the potential of directed induced osteogenetic differentiation of ADSCs in vitro, as to provide theoretical basis for ADSCs as seed cells applying in bone tissue engineering.The second part was the experimental research about PEI mediated gene transfection of rabbit ADSCs. We Observed the toxicity of PEI (13KDa, high branch) in rabbit ADSCs and observed the survival rate of ADSCs under different concentration of PEI and different time points in order to evaluate the safety of PEI as gene carrier for ADSCs. We Observed the microscopic morphology of PEI particles and mixture particles of PEI and DNA in order to evaluate the compatibility of the PEI and DNA plasmid, further discusses the best N/P ratio of PEI combining DNA plasmid (i.e. the number of moles ratio of amino nitrogen of PEI containing and phosphate groups DNA containing). We measured the transfection efficiency of PEI mediated enhanced green fluorescent protein (pEGFP) gene transfection of ADSCs and observed the influence of different N/P ratio on transfection rate in order to discussed the best ratio for obtaining the highest transfection rate, further assess the feasibility and efficiency of using PEI as gene carrier carrier to transfect ADSCs, and look for an ideal transfection carrier for ADSCs gene transfection, and provide theoretical basis for further research about ADSCs osteogenetic differentiation.The third part was experimental study of PEI mediated transfecting BMP-2gene into rabbit ADSCs. BMP-2was recognized as the strongest and the only cytokines which could induce osteogenetic differentiation of stem cells alone. We constructed an eukaryotic expression plasmid pEGFP-N1-BMP-2, and transfect BMP-2gene into ADSCs using PEI as gene carrier, and then detected the gene expression of target cells after transfection, further evaluated the feasibility of PEI mediation BMP-2gene transfection into ADSCs, the stable expression and efficiency of transfection, and the influence of transfection on biological characteristics of ADSCs. We also assessed the efficiency of osteogenetic differentiation through the BMP-2gene transfection into ADSCs mediated by the genetic carrier PEI, in order to further evaluate osteogenetic repair in animal model through the BMP-2gene transfection into ADSCs mediated by the genetic carrier PEI, and discuss the effect of osteogenetic differentiation of rabbit ADSCs induced by BMP-2which released by themselves expression using gene technology in bone tissue engineering seed cells. All that we had done would provide a theoretical basis and experimental evidence for construction of tissue engineering bone and treatment of bone defect and nonunion.Part oneExperimental study of basic biological characteristic and multi-directional induced differentiation of rabbit ADSCsObjective:To investigate the methods of Isolation,culture and basic biological characteristic of ADSCs; to identify its cell phenotype and assess the feasibility as seed cells for bone tissue engineering.Methods:adipose tissue was collected from the neck of adult Japanese white rabbits, to digest the adipose tissue with type I collagenase and obtain primary ADSCs and passage cultured ADSCs when the cells grow to cover about80%of the space.To observe growth situation and morph-ological characteristics of ADSCs with inverted microscope everyday,study the proliferation process and draw the growth curve; cell surface markers CD29, CD34, CD44, CD45were checked by flow cytometry to identify phenotype of ADSCs; the ADSCs were induced to osteogenesis and adipogenesis respectively,the results of induction were assessed through the ALP, alizarin red, VonKossa (calcium nodules) staining and oil red o staining of ADSCs, control group was non-induced.Results:The morphology of ADSCs In vitro culture was spindle-shaped fibroblast-like,proliferation activity,the passage ADSCs morphology was uniform, proliferation was remained strong after several passages, growth curve showed "S" type. Passage3,6of ADSCs are highly expressed CD29, CD44, the positive rate of was more than90%and low expression of CD34, CD45,the positive rate was less than5%, and CD29, CD44increased gradually,CD34, CD45decreased gradually with the passages. Alkaline phosphatase, Alizarin red,von Kossa staining in osteogenic-induction group and oil red o staining in adipogenic-induction were postive and those in the control group were negative.Conclusion:Isolation and culture of Rabbit ADSCs is easy, its proliferation are stable, and express of mesenchymal stem cell-related phenotype,it can be induced to differentiate into osteogenesis and adipogenesis under certain conditions. Bone tissue engineering is expected to become an ideal seed cells.ADSCs may be ideal seed cells for tissue engineering. Part twoPolyethylenimine-mediated gene delivery into rabbit adipose derived stem cellsObjective:To investigate the feasibility of polyethylenimine (PEI,13KDa, branch type) as a non-viral vector-mediated gene transfection to rabbit adipose stem cells. Methods:We examined the cytotoxicity of different concentrations of PEI to adipose stem cell. To transfect enhanced green fluorescent protein gene transfection into adipose stem cells with PEI under different N/P value and obverse the outcome with fluorescence microscope after48hours of transfection and calculate transfection efficiency, with lipofectamine2000transfected as a control group.Results:With the concentrations of PEI at50μg/ml or less, the cytotoxicity of PEI to adipose stem cells was minimal, and the cell viability was more than90%, and cytotoxicity of PEI increased significantly under concentrations of100μg/ml or more; different N/P ratio with different transfection efficiency,when the N/P<6, the transfection efficiency was increased with it; transfection efficiency was the highest when the N/P=6, and thereafter, the transfection efficiency was declined with the ratio of N/P.Conclusion:PEI is a low-cost, safe, practical, high-efficiency gene transfection carrier and it can be used as the gene transfection vector of adipose stem cell. Part threeOsteoblasticdifferentiation of rabbit adipose-derived stem cells transfected by polyethylenimine-mediated bone morphogenetic protein-2gene in vitroObjective:To investigate the expression of human bone morphogenetic protein-2gene transfected to rabbit adipose-derived stem cells by polyethylenimine mediated and assess effect of osteoblastic differentiation of the cells.Methods adipose tissue was collected from the neck of adult Japanese white rabbits, to isolate and culture in vitro to obtain ADSCs, and identify its phenotypic. Polyethylenimine-mediated gene of BMP-2delivery into rabbit adipose derived stem cells, Positive cell clones were selected with G-418. the expression of mRNA of BMP-2was detected with RT-PCR,the protein of BMP-2was detected with ELIS A,osteocalcin and I collagen was detected with Western-blot, Alkaline phosphatase was detected with ALP kit, and stained with alizarin red staining, the untransfected groups as the control groups.Results:ADSCs were successfully isolated from rabbit adipose tissue. Passage3,6of ADSCs are highly expressed CD29, CD44, the positive rate of was more than90%and low expression of CD34, CD45,the positive rate was less than5%, and CD29, CD44increased gradually,CD34, CD45decreased gradually with the passages. BMP-2mRNA transcription and protein expression were found and Alkaline phosphatase, osteocalcin and I collagen increased in transfected ADSCs and Alizarin red staining were postive and those in the control group were negative. Conclusion:It is feasible that polyethylenimine-mediated BMP-2gene transfection into rabbit adipose stem cells, BMP-2gene was effective expression and induce the ADSCs directional osteogenic differentiation after transfected by BMP-2gene.