Comparison Of Stem Cells Derived From Rib Perichondrium,rid Cartilage And Bone Marrow Of Rabbit

There are different point about the origin of mesenchymal stem cells(MSCs), because it has diversitice of source, induction multiplexdifferentiation in vitro, no specific surface features and so on. Manyscholars thought that MSCs come from adult stem cells existed inorganizations and organs through culture and expansion in vitro. ButOrkin prefer that there was no MSCs in the body, just the separated cellswere stimulated, transited or gene reassembly, and became the cells whichhave a characteristics of stem cell, when they were cultured in vitro.Couldadult cells turn into a stem cell? It has been reported that stem cells havebeen separated from cartilage. But there was no stem cells in cartilaginoustissue except cartilaginous vitellarium and arthrodial cartilage. So, it is aquestion that stem cells separated from cartilage were derived from stemcells in cartilage or transformed from cartilage cells?This experiment wasrespectively isolation, culture, amplification stem cells from ribperichondrium、rib cartilage (not vitellarium) and bone marrow of rabbit,to observe whether rib cartilage cells could turn into stem cells and the difference in several sources of stem cells? and to know the mechanism ofthe adult stem cells separation and gain in vitro, and the basis theory of theclinical application of stem cells.PART ⅠIsolation and culture of stem cells derived from ribperichondrium,rid cartilage and bone marrow1Materials and MethodsThe rib cartilage take from New Zealand white rabbits aged3and4weeks except cartilaginous vitellarium, and perichondrium was separatedunder dissecting microscope. Rib perichondrium cells and cartilage cellswere isolated by enzyme digesting method; And bone marrow cells wereisolated from tibias and femurs. Three sources of cells were isolated,cultured and proliferated by adherence screening in vitro, and the stemcells of three sources were obtained respectively. The morphological andgrowth characteristics in each passage cells of three sources were observedtoo.The3passage cells of three sources were culture with1×104in eachhole of culture plate. Three holes of each sources were taken to count cellpopulation and draw their growth curve in10day.2Results The primary generation rib perichondrium cells began to adhesiveafter culture for12h, fusion and assemble as conglobation after culture5-6d, and have a round or polygon form. The2passage cells began toshow a spindle-shape and whirlpool arrangement; and now, the cells hasgrown to the15th generation. The primary generation rib cartilage cellsbegan to adhesive after culture for6h, cell fusion after culture5-6d, andhave a cobblestone appearance, and then, gradually changed asspindle-shape cells from two-passage to there-passage cells. But the cellsappear senescence in the five-passage, and only has grown to the6thgeneration. Bone marrow cells began to be adhesive after culture for4-5h,cell fusion after culture10d. From two-passage, cells show a mainlyspindle-shape and whirlpool arrangement. The cells have grown to the10thgeneration with a good shape. The growth curve of3thpassage cells showsthat their detention period was1-3d, after3d was their log period, after6dwas their platform period.PART Ⅱ Comparison of stem cells derived from rib perichondrium,rid cartilage and bone marrow of rabbit1Materials and Methods1.1The expression of CD29, CD90, CD34and collagen Ⅱ in eachpassage cells of three sources were detected by Immunohistochemical method.1.2The3passage cells of three sources were respectively induced asadipogenic differentiation for14days, or osteogenic differentiation for21days in vitro. Their capacity of osteogenic and adipogenic differentiationwere detected by oil Red O staining and Von Kossa staining.2Results2.1The results of immunocytochemical stain show us that CD29，CD90，CD34were negative in primary and1stpassage cells of ridperichondrium and cartilage; Their2ndpassage cells expressed CD29andCD90, but not CD34. The primary bone marrow cells expressed CD29,CD90and CD34, but not collogenⅡ; And then only expressed CD29and CD90. All the3passage cells of three sources were only expressionCD29and CD90strongly, and their homogenicity achieve to99％. As oneof cell marker of cartilage cells, collogenⅡwas only expressed by theprimary and1stpassage cells of rid cartilage, and stop expressing fromits2ndpassage cells.2.2After adipogenic induction14days in3passage cells of threesources, the form of stem cells derived from perichondrium and bonemarrow became round, but cartilage-derived stem cells became morespindle-shape; The lipid droplets could be seen in cytoplasma of all thesecells after oil Red O staining. After osteogenic induction21days, the stemcells derived from perichondrium and cartilage grew as stratified layer, some black calcified tuberculum minus stained by argent nitrate could beseen on cell surface. The stem cells derived from bone marrow grew assimple layer, but calcified tuberculum minus gathered as bone nodules.Conclusion of whole article1. The stem cells had been isolated from rabbit rib perichondrium andcartilage, and cultured, proliferated successfully by enzyme digest method.BMSCs was isolated by adherence method. Wth the increase of passages,the stem cells derived from rib perichondrium and bone marrow show astronger growth proliferation capacity than stem cells derived from ribcartilage.2.The results detected by immunocytochemical method had shownthat the cells of rib perichondrium and cartilage expressed the samephenotype as BMSCs from the2ndpassage, and strongly expressed in the3rdpassage. It had shown that the three-passage cells derived from ribperichondrium and cartilage has the same phenotype characteristics asMSCs.3. The3rdpassage cells derived from rib perichondrium and cartilagehas the same osteogenic and adipogenic differentiation capacity as BMSCs.It demonstrated that the cells derived from rib perichondrium and cartilagehas the characteristic of multipotent differentiation capability as stemcells.4. The collogenⅡ was only expressed by the primary and1st passage cells rid cartilage. The2ndpassage cells stop expression butexpress CD29and CD90. It indicated that the ripe cartilage cells could bededifferentiation, become stem cells in vitro.5. Although the stem cells derived from rib perichondrium andcartilage show the same immunophenotype and osteogenic and adipogenicdifferentiation capacity as BMSCs, the growth proliferation capacity of ribperichondrium-derived stem cells were stronger than those of stem cellswhich derived from rib cartilage, and after dedifferentiation, transformedas stem cells. So, when we obtained stem cells from the cartilages, the ribperichondrium should be reserved as far as possible.