| Part I The construction of lentiviral vector, BMSC separation and culture, gene transfectionObjective The short hairpin RNA (shRNA) eukaryotic expression vector targeting of the PPARy gene was constructed and transfected into cultured rat bone marrow stem cells (BMSC) via lentiviral expression vector, which also contains "Tet-On" expression system. The inhibition effects on PPARy gene and the ability to undergo osteogenic and adipogenic differentiation were determined under the different Dox concentration.Methods The PPARy gene of mouse was designed from the mouse PPARy mRNA sequence, which also contains "Tet-On" expression system and the gene of red fluorescence protein (RFP). The plasmid vector was isolated, purified and transfected into rat BMSC. To observe the expression of PPARy genes and RFP cultured in vitro. The expression of PPARy gene and protein, the adipogenic factors adipocyte determination and differentiation-dependent factor1(ADD1), the osteogenic genes encoding collagen I were measured by RT-PCR and Western blot assay following PPARy silencing. To observe tBMSC after the osteoinductive culture under the different Dox concentration by detecting changes of the activity of alkaline phosphatase(ALP) and the amount of calcium deposition as measured by Alizarin red Sstaining, and the lipid droplet count as measured by Oil red O staining.Results The shRNA target sequence (GTCTGCTGATCTGCGAGCC) was successful designed and cloned into "Tet-On" ukaryotic expression vector. After measuring virus titer (3.6x10E8TU/ml), BMSC were transfected and the RFP fluorescence intensity was observed under fluorescence microscope. RT-PCR and Western blotting showed that the expression level of PPARy was significantly inhibited by PPARy-shRNA compared with controls, the inhibition rate was36%at24h (P<0.01) and53%at48h (P<0.01). The mRNA levels of ADD1in tBMSC were32%(p<0.05) at3d and75%(p<0.01) at7d when compared with BMSC. The mRNA levels of collagen I in tBMSC were37%(p<0.05) at3d and66%(p<0.01) at7d when compared with BMSC. The tBMSC induced to differentiate into adipocytes for9days, the activity of ALP increased with the increase of concentration of doxycycline hyclate (Dox) between2μg/ml and8μg/ml, and decreased from10μg/ml. The OD values of the destained solution of Oil red O from tBMSC was61%when compared with BMSC (p<0.01) after14days of adipogenic induction.Conclusion The efficiencies were high with eukaryotic expression vector in tBMSC, the down regulation of PPARy by shRNA inhibited adipogenic differentiation and promoted osteogenic differentiation. The tBMSC had showed active proliferative capacity and inheritable stability of PPARy silencing after generation, the inhibition ratio relianced on the Dox concentration, the optimal Dox concentration for promote osteogenesis was8μg/ml. Part IIThe preparation of BCP scaffold coated with HA/PLLA nanomposites, the preparation of microspheres with Dox and loade the microspheres on the BCP scaffoldsObjective The purpose of this part is preparing the linear PLGA-mPEG microspheres contain Dox, the micropore was loaded on the pore wall of BCP scaffold coated with HA/PLLA nanomposites to produce a biomineralized materials with slow release system for bone tissue engineering.Methods The round disks2mm×5mm(R)multihole BCP scaffolds coated with two layer of HA/PLLA nanocomposites were prepared by the method of organic foam plastic. The linear PLGA-mPEG (PLGA-mPEG, LA/GA=9:1) microspheres loaded with Dox was prepared by multiple emulsion method. The microspheres were loaded on the BCP scaffolds coated with HA/PLLA. The structure and morphology of BCP/Dox scaffolds was analyzed by means of metallographic microscope (SEM) and Fourier transform infrared spectroscopy (FTIR). The encapsulation rate, drug content and the release rate of Dox were calculated in Phosphate Buffer solution (PBS) in vitro at37℃, and the results were statistically analyzed.Results The drug content was0.5%, the encapsulation rate was24.7%.The size of the microspheres was uniform and between15μm and20μm. The microspheres could be easy loaded on the BCP scaffolds with pore size between300μm and600μm. The drug release experiments in vitro showed that there was burst release phenomenon, especially in the first16h, the release percentage was32.6%in the first8h and65.5%in the first7d, and the stage of slow release started from7d, and continued for60d.Conclusion The pore size of BCP/Dox scaffolds between300μm and600μm, porosity between90.8%and92.3%, and compressive strength between4.8MPa and 5.73MPa, the scafford can meet essential requirements for bone tissue engineering and regeneration. The microspheres loaded with Dox had good slow release effect for a long time. Part Ⅲ Repair of rat bone defects by BCP/Dox scaffolds combine with tBMSC cellsObjective Making rat cranial bone defects model, and implanting the BCP/Dox scaffolds combined with tBMSC cells into the defect site. Dox releasing from BCP/Dox scaffolds regulated tBMSC promoted osteogenic differentiation by controlling release and target delivery, to explore a new method for the therapy of bone defect.Methods The effect of BCP/Dox scaffolds on tBMSC and BMSC adhesion was measured with cell counting; the proliferation was detected by MTT at1d,3d and5d; the osteogenic differentiation was detected by the qualitative analysis of ALP after the osteoinductive culture9d, and by alizarin staining to count calcium depositions after the osteoinductive culture11d. The adipogenic differentiation was detected by oil red staining after14d culture. The biocompatibility of BCP/Dox scaffolds in vivo was measured by the acute toxicity experiment, the sensitization test and the hemolysis test. The ectopic osteogenesis was investigated in the muscle of rat by the histological method, total30SD rats of6w, making a muscle bag in the medial posterior thigh of right side models and implanting a BCP/Dox scaffolds,6rats was sacrificed respectively in1d,7d,14d,21d and28d for HE to study the ectopic osteogenesis potential. Divided the45SD rats of6w in age into control group (9rats), BMSC+BCP/Dox group (18rats), and tBMSC+BCP/Dox group (18rats) random in each group. Making rat cranial bone defects model, and implanting the BCP/Dox scaffolds combined with tBMSC or BMSC into the defect site for the BMSC+BCP/Dox group and tBMSC+BCP/Dox groups. After operation, rats were sacrificed respectively in4,8and12weeks, to measure the bone mass by the X-ray and Micro-CT.Results The adherence rate of tBMSC on the surface of the BCP/Dox scaffolds was lower than BMSC at6h and24h, there were no significant differences between the two groups(p>0.05), and the adhesion rate was more than70%in the two groups. There was no significant differences for the proliferation rate at Id and3d, tBMSC+BCP/Dox group smaller than those in tBMSC+BCP/Dox group at5d, there were no significant differences between the two groups (p>0.05). The SEM showed that the adhesion and the proliferation rate of tBMSC on the surface of BCP/Dox scaffolds, and there were extracellular matrix and microspheres in the space surrounding the cells.The ALP activity was higher in the tBMSC+BCP/Dox group than other two groups (p<0.01) at6d and9d, and BMSC+BCP/Dox group>BMSC group (p<0.01), tBMSC+BCP/Dox group> BMSC+BCP/Dox group (p<0.01). The alizarin staining showed that the calcium nodule numbers were highest in tBMSC+BCP/Dox group and lowest in BMSC group. The quantitative analysis of Oil Red Ostaining extraction showed that tBMSC+BCP/Dox group less than BMSC groups (p<0.01) and BMSC+BCP/Dox group less than BMSC groups (p<0.05). X-ray showed that the sharp edges of defect in all groups at4w, and blurred edges of deceet in two BCP/Dox groups at8w, and bone fusion in tBMSC+BCP/Dox group at12w; The gray analysis showed that tBMSC+BCP/Dox group was higher than BMSC+BCP/Dox group (p<0.01), and BMSC+BCP/Dox group was higher than BCP/Dox group (p<0.05). The Micro-CT analysis showed that the bone mass were highest in tBMSC+BCP/Dox group and lowest in BCP/Dox group, there were statistical significance was found between groups (p<0.01).Conclusion The BCP/Dox scaffolds possessed good biocompatibility and high bone inductive potentiality, it can be used to repair bone defect combined with tBMSC cells. |
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