| Part ? Purification and detection of mice articular cartilage-derived progenitor cellsObjective:ACPCs were isolated and cultured from mice articular cartilage.The morphology of ACPCs was observed and the proliferation ability,differentiation ability and surface markers were identified,which explored the the cellular characteristics of mice ACPCs to provide preparation for later ACPCs-based cartilage tissue engineering.Methods:The cartilage tissue was obtained from the joints of 5-day-old male C57BL/6 mice under sterile condition.Cell suspension was obtained by overnight digestion with type IV collagenase.ACPCs were obtained by using differential adhesion to fibronectin.The expression of surface markers was determined by flow cytometric analysis.The cell morphology was observed by inverted microscope when the cell reached 80%.The proliferation ability was evaluated by clonal formation unit test.The mult-differentiation ability was detected by three-lineage induction.Results:Mice ACPCs were found to be short spindle-shaped with middle width and slender sides by using inverted microscopy.The morphology of ACPCs was similar to that of mesenchymal cells.The expressions of CD29 and CD73 were(89.2±1.53)%and(81.1±0.84)%,CD90 and CD105 were(24.3±1.11)%and(41.6±0.62)%,Notch-1 was(12.0±1.05)%,CD31,CD44 and CD45 were low or no expression.Clonal formation unit test showed that mice ACPCs could form multiple single cell clone clusters.Calcium nodules were found in the six-well plate under osteogenic induction;a large number of lipid droplets were seen under adipogenic induction,and spontaneous lipid droplets were also seen without adipogenic induction;Cartilage lacunaes with extracellular matrix surrounded were observed in HE staining of pellet under chondrogenic induction.Conclusion:ACPCs were successfully obtained from mouse articular cartilage by using differential adhesion to fibronectin,which has three-lineage differentiation potential.Part ? The study on proliferation,osteogenic and chondrogenic differentiation of mice articular cartilage-derived progenitor cellsObjective:To assess the difference of proliferation ability,osteogenic and chondrogenic differentiation ability of mice ACPCs during cell expansion and find an effective proliferation and differentiation induction scheme of mice ACPCs.Methods:Different generations(P0,P1,P2)of mice ACPCs and different bFGF concentrations(0 ng/mL,2 ng/mL and,ng/mL)of P1 mice ACPCs were used.The cell morphology was observed by inverted microscope.Clonal formation unit test and cell proliferation experiments were used to compare the difference in proliferation ability.Alizarin red S staining and RT-PCR of osteogenic related genes(Osteocalcin,AKP,Runx2)were used to compare the difference in osteogenic differentiation potential.The HE staining,collagen ? immunohistochemical staining and RT-PCR of chondrogenic-related genes(COMP,SOX9,Aggrecan,COL-?)were ued to compare the difference in chondrogenic differentiation potential.Results:(1)There was no obvious change in morphology during cell expansion.The cell density decreased with the increase of generations.The number of single cell clone clusters in clonal formation unit test and OD value of cell proliferation experiment of mice ACPCs decreased during cell expansion.The results of alizarin red S staining and RT-PCR showed that the osteogenic potential of mice ACPCs decreased during cell expansion.The results of HE staining,collagen ? immunohistochemical staining and RT-PCR showed that the chondrogenic potential of mice ACPCs remained unchanged during cell expansion.(2)The cell morphology of mice ACPCs changed from short spindle cells to long spindle cells with the increase of bFGF concentration.The cell density was the highest when the concentration of bFGF was 2 ng/mL.The OD value and clonal formation unit test showed that ACPCs had the strongest proliferation ability at 2 ng/mL bFGF concentration.The results of alizarin red S and RT-PCR showed that the osteogenic potential of ACPCs decreased with the increase of bFGF concentration.HE staining,collagen ? immunohistochemical staining and RT-PCR showed that ACPCs had the strongest chondrogenic potential at 2 ng/mL bFGF concentration.Conclusion:The proliferation and osteogenic differentiation potential of mice ACPCs decreased during cell expansion,while the chondrogenic differentiation ability did not change significantly.The culture condition of a-MEM containing 2ng/mL bFGF was a relatively suitable condition for the proliferation and differentation induction of mice ACPCs.Part ? The study on the stability of mice ACPCs and anti-angiogenesis in vivoObjective:To investigate the stability of mice ACPCs-based pellet in vivo and the anti-angiogenesis effect of Axitinib.Methods:ACPCs of P2 mice cultured in a-MEM complete medium containing 2 ng/mL bFGF were treated in the following ways:(1)Chondrogenic induction group(Group A)contained the pellets cultured in chondrogenic induction medium for 3 weeks.The pellets in control group(Group B)were cultured in high glucose standard medium for 3 weeks;(2)Geltin-PCL Electrospinning containing Axitinib was used in experimental group(Group C).The material without Axitinib was the control group(Group D).The ACPCs-based engineered cartilage were constructed by using "Sandwich Model" and chondrogenic induced in vitro for 3 weeks.All groups were implanted subcutaneously in the back of BALB/C nude mice.2,4 and 6 weeks after operation,the nude mice in Group A and Group B were killed and the pellet was obtained.The mice in Group C and Group D were killed at 6 weeks after operation to obtain the ACPCs-based engineered cartilage.HE,saffron O-fast green staining,type ? collagen,type ? collagen and VEGF-A immunohistochemical staining were performed.Results:(1)The results of HE staining and safranine O-fast green staining showed that cartilage lacunae disappeared and cartilage ossification began to appear in Group B at 2 weeks after implantation.More and more ossification occurred in both Group A and B with the prolongation of implantation time in vivo.There was no ossification in Group A at 2 weeks after operation.The cartilage ossification of Group A occurred at 6 weeks after implantation.Immunohistochemical staining of type ? collagen showed,with the increasing time in vivo,the brown stained area of pellet decreased gradually;Immunohistochemical staining of type X collagen showed that the brown stained area of pellet was positively correlated with implantation time in vivo;Immunohistochemical staining of VEGF-A showed that the brown stained area of pellet increased with the prolongation of implantation timeand was aggregated around calcified tissue.(2)The results of HE staining and safranine O-fast green staining showed that the ACPCs-based engineered cartilage in Group D ossification,while the experimental group had no ossification at 6 weeks after operation.The immunohistochemical staining of collagen ? showed that Group C had strongly positive areas,while Group D had no positive area;The immunohistochemical staining of collagen X had positive areas both in Group C and Group D;The immunohistochemical staining of VEGF-A showed that there were positive areas around calcification in Group D and no positive area in the Group C.Conclusion:The cartilage ossification would occur in vivo even the pellets had formed the stable cartilage tissue morphology in vitro,which may be related to angiogenesis.Axitinib,an inhibitor of VEGF-A receptor,can inhibit the ossification of ACPCs-based engineered cartilage in vivo. |
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