| ObjectivePart one Using ion self-assembly and electrostatic complexation,a polyelectrolyte graded film with similar structure to hyaline cartilage was prepared using chitosan and sodium alginate as raw materials,and its effect on cell proliferation and differentiation was studied.Part two The effect of polyelectrolyte graded film with biomimetic structure on the repair of cartilage injury was studied through in vivo experiments.MethodsPart one Chitosan/sodium alginate graded film with biomimetic structure was prepared by direct solution reaction method,and the same composition film material without biomimetic structure was prepared by controlling the reaction temperature;The general appearance,internal structure,porosity,swelling rate and degradation rate of the film material were characterized;After inoculating rabbit cartilage stem cells on the membrane material,the effect of the membrane on cell proliferation was studied by CCK-8 experiment and dying cell staining;After the rat bone marrow mesenchymal stem cells were inoculated on the membrane material,they were induced to differentiate into cartilage.The effect of the membrane material on the differentiation of stem cells was studied by Alcian blue staining and RT-qPCR.Part two 40 3-month-old male New Zealand white rabbits were randomly divided into4 groups: sham operation group,blank control group,random material group and regular material group;After the rabbit knee full-thickness cartilage injury model was established,the treatment was carried out according to the treatment methods of each group.On the first day,the fourth week and the eighth week after the successful modeling,the animals in each group were examined by magnetic resonance imaging,and the images were scored by the whole organ magnetic resonance imaging scoring system(WORMS);At the 4th and 8th weeks after modeling,half of the animals(5 animals,10 sides)in each group were killed,and gross specimens of femoral condyles were obtained,and then observed and scored using the International Association for Prosthetic Cartilage(ICRS)macroscopic scoring standard;Then the gross specimens were paraffin sectioned and stained with HE and safranine O-solid green,and then scored with ICRS histological scoring standard;CollagenⅡ and ACAN immunohistochemical staining were performed,and their positive expression was analyzed quantitatively.ResultsPart one The appearance of the film material is yellow-brown film.The film material prepared at 60 ℃ has a structure similar to that of transparent cartilage tissue.We call it regular material,while the film material prepared at 25 ℃ does not have this structure.We call it random material;The swelling rate of regular materials was(360.60 ± 23.33)%,which was higher than that of random materials(270.00 ± 35.67)%(P<0.001);The porosity of regular materials is(85.32 ± 2.91)%,which is higher than that of random materials(61.23± 3.97)%(P<0.001);The degradation rate of regular materials was higher than that of random materials,but there was no significant difference between them at each time point(P>0.05).The survival rate of cells on the two kinds of film materials is more than 95%,which has no adverse effect on cell proliferation.When the stem cells inoculated into the material induced differentiation to the 14 th day,the blue-stained area of the regular material group was(49.67 ± 4.22)%,which was larger than that of the random material group(35.56± 4.10)%(P<0.05);At the 28 th day of differentiation,the blue-stained area of the regular material group was(94.67 ± 5.21)%,which was higher than that of the random material group(89.25 ± 4.12)%(P<0.05).RT-qPCR experiment showed that the mRNA expression of COL2A1 and ACAN in the group of random materials was higher than that in the group of random materials at different time points of induced differentiation(P<0.05).Part two At 4 weeks after operation,the WORMS score in the blank control group was 5.40 ± 0.95 points,which was higher than 3.89 ± 0.56 points in the random material group(P<0.05)and 2.36 ± 0.45 points in the regular material group(P<0.001);At 8 weeks after operation,the WORMS score of the blank control group was 2.33 ± 0.41 points,which was greater than 1.25 ± 0.35 points of the random material group(P<0.05)and 0.50 ± 0.22 points of the regular material group(P<0.001).At 4 weeks,the ICRS macro score in the blank control group was 2.76 ± 0.56 points,which was lower than 4.12 ± 0.49 points in the random material group(P<0.01)and 6.25 ± 0.68 points in the regular material group(P<0.001),;At 8 weeks after operation,the ICRS score in the blank control group was 5.98± 0.74,lower than 8.01 ± 0.87 in the random material group(P<0.05)and 11.05 ± 0.55 in the regular material group(P<0.001).At 4 weeks after operation,the ICRS histological score in the blank control group was 2.55 ± 0.25 points,less than 5.75 ± 0.64 points in the random material group(P<0.01),less than 7.80 ± 0.83 points in the regular material group(P<0.001);At 8 weeks after operation,the score of blank control group was 7.20 ± 0.40 points,which was lower than 10.48 ± 0.89 points of random material group(P<0.05)and16.50 ± 1.20 points of regular material group(P<0.001).Immunohistochemical quantitative analysis showed that at the 4th week after operation,the AOD value of Collagen Ⅱ staining in the regular material group was significantly higher than that in the blank control group and the random material group(P<0.05),and ACAN staining also showed the same result;At the 8th week after operation,the AOD value of Collagen Ⅱstaining in the regular material group was significantly higher than that in the blank control group(P<0.001)and the random material group(P<0.05),while in ACAN staining,the AOD value of the regular material group and the random material group had no statistical difference(P>0.05).ConclusionsPart one The chitosan/sodium alginate grading film with bionic structure successfully mimics the structure of hyaline cartilage,has no negative effect on cell proliferation,and can promote the differentiation of rat bone marrow mesenchymal stem cells into chondrocytes.Part two The chitosan/sodium alginate graded film with bionic structure promotes the repair of cartilage damage,and the bionic structure plays an active role in the repair process. |
