| OBJECTIVELacking of the integrated and standard method of bone marrow mesenchymal stem cells (MSCs) culturing, identifying, inducing and differientiating MSCs into chondrocytes, in this study, MSCs were cultured and expanded in vitro to observe the general morphological structure of MSCs. MSCs were detected by flow cytometry, immunofluorescence and MSCs differientiating functional method. MSCs were induced and differentiated into chondrocytes by TGF-β1and IGF-1, Comparing the amount of the Ⅱ type collagen, when using alone and a combination of the two factors in different induced time. The tiny and ultrastructure changes of MSCs before and after induction were studied by light microscope and electron microscope, detecting the effect of TGF-β1and IGF-1on the proliferation of MSCs. This study provided the experimental base and technical feasibility for the further application of MSCs as tissue engineering cartilage seed cell and construction of tissue-engineering tracheal cartilage.METHODS1MSCs acquisition, culture, purification and detection1.1MSCs acquisition, culture, purification①Cervical dislocation executed rats. The bone marrow was obtained after Rat’s femur and tibia were separated under aseptic condition.②Impurities cells were removed by renewing liquid and cell passage.1.2MSCs detection①Morphological identification MSCs were detected by immunofluorescence②Phenotype identification MSCs were detected by flow cytometry③functional identification MSCs were differentiated into chondrocytes.1.3MTT detection The effect of TGF-β1and IGF-1on the MSCs proliferation was detected by MTT.1.4The morphological changes of MSCs before induction were observed by scaning electron microscope.1.5The morphological changes of MSCs before induction were observed by transmission electron microscope.2Induction and differentiation of MSCs into chondrocytes2.1Inducing in vitro and grouping The experiment included4groups (A group,50ng/ml IGF-1and10ng/ml TGF-β1; B group,10ng/ml TGF-β1; C group,50ng/ml IGF-1and control group without growth factor). A, B and C group in DMEM-H mediem were added vitamin C50μg/ml, insulin6.25μg/ml and dexamethasone51.6ng/ml as basic induced liquid. The fourth MSCs generations were cultured in six orifices by1×105/ml, covering glass in the orifices for cellular climbing piece.2.2The morphological changes of MSCs after induction were observed by scaning electron microscope.2.3The morphological changes of MSCs after induction were observed by transmission electron microscope.2.4Detecting after induction and differentiation The MSCs induced and differentiated were detected by immunofluorescence, RT-PCR detection and immuncytochemical staining.RESULTS1Light microscopy observation Most cells showed fusiform, triangle and a little showed rounded after removed non-adherent cells in24hours. The thired MSCs form were uniformed and impuritied cells were rare. The cellular shape changed and showed triangle, star and polygon after induced for7days. The most cells showed triangle, star and polygon and multiple processes were visible after induced for21days.2MSCs detection①the experimental cells highly expressed CD90and CD29and rarely expressed CD34and CD45by flow cytometry and immunofluorescence.②The experimental cells expressed CD90and CD29over99%and didn’t expressed CD34and CD45.3MTT detection The proliferation activity in group A, group B and group C were higher than control group(P<0.05).TGF-β1and IGF-1could enhance MSCs proliferation activity.4Observations by scanning electron microscope MSCs showed long fusiform, tile tretching and protuberance was visible. After induced, cell volume increased and showed polygon, star and triangle. Several protuberances were visible.5Observations by transmission electron microscope A MSCs nucleus and a nucleolus were visible by transmission electron microscope before induced. Mitochondrias also were visible in cytoplasm before induced. The nucleus augment and the nucleolus increased after induced for7days. The mitochondria, rough endoplasmic reticulum and cavitation increased after14days. Microvilli and lipid drops were visible. Cavition and lipid drops increased after21days. Several small myelin bodies were visible.6Detecting after inducing and differentiation6.1Immunofluorescence In A group and B group the cytoplasm showed palm red and highly expressed collagen type Ⅱ. Group C and control group were negative.6.2RT-PCR detection RT-PCR result showed collagen type Ⅱ amplification bands were seen and showed positive. Group C and control group were negative.6.3Immuncytochemical staining Collagen type Ⅱ immuncytochemical staining of Group A and group B were positive and the cytoplasm showed palm red. Group C and control group were negative. The expression of group A Collagen type Ⅱ was significantly higher than group B and the expression of collagen type Ⅱ was gradually increased.CONCLUSIONIn this study, a large number of high purified MSCs were cultured in the method of whole bone marrow adeherent culture, and met the demand to construct tissue-engineered trachea as the seed cell, so the method of whole bone marrow adeherent culture was recommended. Flow cytometry, immunofluorescence and MSCs differientiating functional method were an ideal method to detect MSCs at present. TGF-β1and IGF-1could promote proliferation of MSCs. The tiny and ultrastructure changes of chondrocytes differentiated from MSCs indicated that the cells were active in differentiation and metabolism. Using TGF-β1and IGF-1might acquire the best inductive effect in chondrogenic differentiation of MSCs in vitro. So as to construct the better completed, standarded model of MSCs culture and directional differentiation into chondrocytes. This study provided the experimental base and technical feasibility for the further application of tissue-engineering technology to repaire tracheal cartilage defection. |
PDF Full Text Request