Effect Of Light Curing Injectable Locust Bean Gum Hydrogel On BMSCs Chondrogenesis

Purposes:This topic aims to explore and study the locust bean gum hydrogel modified by methacrylic anhydride(MA)in inducing the proliferation and chondrogenic differentiation of bone marrow mesenchymal stem cells(BMSCs)and the role of repairing defective cartilage tissue.Methods:Fisrst of all,light-curing and injectable modification of LBG-MA and its characterizations:dissolve 1.0g of LBG in 100ml of deionized water,then add 3ml of MA solution to the LBG solution when the water bath is stirred and heated to 50℃,the LBG aqueous solution was adjusted to neutral and the reaction was heated for 6 hours.Dialysis,purification,freeze-drying,the final product is the modified product.The FTIR and ~1H-NMR were used to analyze whether the grafting modification of LBG was successful;The pore size and morphology of LBG-MA were observed by SEM;The mechanical properties,swelling and degradation properties of hydrogel were analyzed by measuring the mechanical properties,swelling rate and degradation rate of LBG-MA.Next,light-curing injectable LBG-MA inducing BMSCs into cartilage differentiation in vitro:the third-generation BMSCs were planted on the surface of LBG-MA with different concentrations.The concentration of the least toxic LBG-MA was selected by MTT detection and mixed with the third-generation BMSCs to form a hydrogel/cell complex;After 7,14 and 21 days of culture,the survival and proliferation of BMSCs were observed by calcein/PI staining;The morphology of chondrogenic differentiation was observed by HE staining;GAG/DNA was used to detect the secretion of chondrogenic differentiation index;q RT-PCR was used to detect the expression of cartilage related characteristic genes to verify the ability of chondrogenic differentiation.Last but not least,validation of light cured and injectable LBG-MA BMSCs for regeneration and repair of cartilage defects:the model of cartilage defect was established,the cells and 2%,4%light cured LBG-MA/cell complexes,10%light cured LBG-MA/cell complex(group B,LBG-MA2 group,LBG-MA4group,Gel-MA10 group)were implanted into the cartilage defect site.At the fourth week after operation,the samples were collected and photographed,and the gross score of the joint was further evaluated to observe the regeneration and repair of the defect cartilage.Finally,histological staining and histological score were used to observe the repair of cartilage defects.Results:It was verified by FTIR and ~1H-NMR that the modification of LBG by MA was successful,and the LBG-MA has the property of curing into gum under ultraviolet light irradiation.The swelling performance test results showed that the LBG-MA basically reached the swelling equilibrium within 6hours,and the greater the concentration of the hydrogel,the lower the swelling rate.The results of the degradation performance test showed that the greater the concentration of the hydrogel,the lower the degradation rate.The Young’s modulus results showed that the greater the concentration of the hydrogel,the greater the Young’s modulus.Compression performance testing showed that the greater the concentration of the hydrogel,the greater the corresponding compressive stress when the hydrogel breaks.The rheological performance test showed that the change of G’with the percentage of LBG-MA was consistent with the measured value of compressive modulus.SEM results showed that the has good through-hole characteristics and uniform pore size.The change of hydrogel concentration can make LBG-MA possess good pore size and suitable Young’s modulus,thus making the structure consistent with the structure of the extracellular matrix.The results of calcein/PI staining showed that the number of cells in the LBG-MA2 group increased with the increase of culture time,and the survival rate was higher.The results of HE staining showed that obvious cartilage lacunas could be observed in the LBG-MA2 group.GAG/DNA test results proved that the LBG-MA2 group had the same secretion amount of specific extracellular matrix as chondrocytes.The results of q RT-PCR showed that the expression of cartilage-specific marker genes in the LBG-MA2 group was higher.The results of in vivo experiments showed that the new cartilage tissue in the LBG-MA2 group was similar to the adjacent normal cartilage tissue,and the repair effect was better.The histological staining results are consistent with the general observation results,verifying the effect of photocurable LBG-MA combined with BMSCs in repairing cartilage defects.Conclusions:(1)The LBG-MA can be cured under UV irradiation,with uniform pore size,good swelling property,degradation property and mechanical strength;(2)Light cured LBG-MA has good ability to induce chondrogenic differentiation of BMSCs in vitro;(3)Light cured LBG-MA composite BMSCs has good effect on regeneration and repair of defective cartilage.