| Objective:To investigate the ability of Ad-TGF?1 infected BMSCs adhering to 3D printed PLA scaffold to repair rabbit knee cartilage defects,and to further study the feasibility of 3D printing technique combined with Ad-TGF?1 infected BMSCs to repair articular cartilage defects.Methods:1.BMSCs isolation and culture: two-month-old New Zealand white rabbits were killed by water flooding.Bone marrow fluid was taken from bilateral tibia with 10 ml syringe and added to the centrifuge tube containing Percoll separation solution.BMSCs was isolated and cultured by 3000r/min centrifugation 30 min.The liquid was changed every 3 days and its growth was observed.The third generation cells(P3)were frozen at-80 ℃.2.The amplified Ad-TGF?1 was used to infect BMSCs cells.The TGF?1gene was introduced into BMSCs cells and cryopreserved at-80 ℃.3.The rabbit knee cartilage defect model was constructed,and the rabbit knee cartilage defect was scanned by CT.The cartilage defect scaffold with a diameter of 100 μm was designed by 3D technology.The cartilage defect scaffold was printed with PLA as the material,and the printed PLA scaffold was immersed into the Na OH solution scaffold of 2mol/L to modify the surface.Set aside after ultraviolet disinfection.4.BMSCs carrying Ad-TGFβ1 was inoculated into disinfected PLA scaffold and co-cultured to construct Ad-TGF β 1 mediated BMSCs-PLA scaffold complex.Five days later,the structure of the scaffold complex and cell adhesion were observed by scanning electron microscope.5.36 New Zealand white rabbits were randomly divided into three groups:blank group,PLA group and PLA/BMSCs group.The blank group was simple cartilage defect group.PLA stent was implanted only in PLA group,and BMSCs-PLA stent composite was implanted in PLA/BMSCs group.After treatment,the animals were fed in a single cage.6.At the 8th and 12 th week,the newborn tissues of knee joint defects of each group were stained with HE and fuchsin O respectively,and the new tissues were observed under inverted phase contrast microscope.7.Westernblot method was used to semi-quantitatively detect the expression of type II collagen in the newborn tissue of knee joint defects in each group at the 8th and 12 th week.Results: 1.BMSCs were isolated from rabbit bone marrow and subcultured to P3.2.The amplified ad TGF β 1 can infect BMSCs successfully.3.The PLA scaffolds with 100 μm aperture were successfully printed by 3D printing technology,and the BMSCs with AD TGF β 1 could adhere to them to form BMSCs PLA scaffolds complex.4.There was no obvious inflammatory reaction in the knee joint of the three groups of experimental animals.In the PLA / BMSCs group,pink new tissue similar to hyaline cartilage could be seen at the cartilage defect 8 weeks after operation,and its surface was smooth without depression.In the PLA group,rough pink new tissue was found on the surface of the defect,and its elasticity and hardness were worse than those in the PLA / BMSCs group;in the blank group,cavity collapse was found on the cartilage defect,and a little white new tissue was found around the defect,with clear boundary.At 12 weeks,there were white new tissue on the cartilage defect of PLA / BMSCs group,the surface was smooth without depression,closely combined with the adjacent normal tissue,the elasticity and hardness of the new tissue were not significantly different from that of the adjacent normal cartilage tissue;there were some milky white new tissue on the defect of PLA group,the surface was slightly rough and the elasticity and hardness of the new tissue were worse than that of PLA /BMSCs group.In the blank group,the cartilage defect was obviously depressed and the boundary with surrounding tissue was clear.5.At 8 weeks after the operation,HE staining was performed on PLA / BMSCs group.In the PLA group,new granulation tissue were found in the defects and the boundary was clear.In the blank group,there was a little fibrous tissue hyperplasia in the defect.At 12 weeks after the operation,the defects of PLA / BMSCs group were filled with new tissue,columnar arrangement of hyaline cartilage and a large number of cartilage lacunae.In the PLA group,there were irregular new tissues and a small number of cartilage lacunae which were composed of hyaline cartilage under the microscope.No new chondrocytes were found in the blank group.6.At 8 weeks after operation,the new hyaline chondrocytes stained with safranine were filled in the defects of PLA / BMSCs group,which were arranged vertically on the surface and horizontally in the deep.In the PLA group,there were some irregular new hyaline chondrocytes in the defect,the staining was lighter than that in the PLA /BMSCs group,and no new chondrocytes were found in the blank group.At 12 weeks after operation,the defects of PLA / BMSCs group were completely filled with new hyaline chondroid cells with deep staining.In the PLA group,a large number of new hyaline chondrocytes with irregular arrangement were observed,and the staining was lighter than that in the PLA / BMSCs group.In the blank group,there were some fibrous tissue hyperplasia and no obvious new chondrocytes.7.After the operation,the expression of col-ii protein in the new tissue of articular cartilage defect area of each group was detected by Western blot.It was found that the expression of col-ii protein in PLA / BMSCs group was significantly higher than that in PLA group and control group at the 8th and 12 th weeks(P < 0.05).The expression of col-ii protein in PLA group was also higher than that in control group(P < 0.05).8.Wakitani score was used to evaluate the repair effect of cartilage defects in each group at 8 and 12 weeks after operation.The results showed that the difference of wakitani score in blank group,PLA group and PLA / BMSCs group was 10.830 ±1.170,8.330 ± 0.817,6.500 ± 0.547,F = 36.500,P = 0.000 < 0.05,respectively,which indicated that there was statistical significance in the three groups.At 12 weeks after operation,the wakitani scores of blank group,PLA group and PLA / BMSCs group were 9.670 ± 1.033,7.500 ± 1.265,4.670 ± 1.211,F= 28.230,P = 0.000< 0.05,which indicated that the wakitani scores of PLA / BMSCs group were significantly lower than those of the other two groups(P < 0.05).Conclusion: BMSCs infection with Ad-TGF?1 can form BMSCs-PLA scaffold complex on PLA scaffold,which is beneficial to cartilage regeneration and repair after implantation into rabbit knee cartilage defect area. |
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