| Objective:This study was conducted to study the effect of microbial-derived extracellular mucopolysaccharide polysaccharide pullulan on the proliferation and chondrogenic differentiation of bone marrow mesenchymal stem cells and the repair of cartilage defects in vivo.Methods:The method of the first part:Photocurable modification and characterization of pullulan hydrogel:Dissolve 5.0 g of pullulan in 50 ml of distilled water,stir to dissolve,stir and heat in a water bath at 50?.When the temperature reaches 50?,slowly add 5 ml of Methacrylic anhydride,stir while stirring,use 5 moL/L The pH of the solution was adjusted to neutral by NaOH solution,and heated at 50? for 8 hours in a water bath.Dialysis,purification,lyophilization of the resulting modified product.Fourier transform infrared spectroscopy was used to analyze whether the grafting was successful and further verified by 1HNMR;The morphology and pore diameter of the photocopiable modified pullulan hydrogel were observed by scanning electron microscopy;The Young's modulus and the swelling ratio were measured,and the mechanical strength and swelling property of the hydrogel were analyzed.The method of the second part:Induction of cartilage differentiation of bone marrow mesenchyme stem cells by photocopiable modified pullulan hydrogel in vitro:SD rat bone marrow mesenchyme stem cells were extracted and cultured in vitro,and the third-generation was planted on a series of different concentrations of pullulan hydrogel for 7 days.CCK8 was utilized to detect different concentrations of pullulan hydrogel.The effect of bone marrow mesenchyme stem cell toxicity,the least toxic concentration is selected for the preparation of hydrogels mixed with third-generation cells to form hydrogels/cell complexes;Cell viability and proliferation were observed by calcein/PI staining after 7 days,14 days and 21 days of culture.HE staining was utilized to observe the cartilage differentiation morphology,and the cartilage differentiation index(glycosaminoglycan)was observed by Safranin O staining and GAG/DNA detection.The secretion amount of COL2a1 was observed by immunofluorescence staining of cartilage marker protein type ? collagen;cartilage-related characteristic genes COL2a1,SOX9,ACAN,COL1a1,COL10a1 were detected by RT-PCR after 7 days,14 days and 21 days of culture.The expression level checked as the ability to differentiate into cartilage.The method of the third part:Verification of the repair and regeneration of cartilage defects in vivo by photocopiable modified pullulan hydrogel composite stem cells:The SD rat articular cartilage defect model was constructed,and different groups of cells and photocopiable modified pullulan hydrogel/cell complex(B group,BS group,BC group,BP group)were implanted into the defect site.At the 4th week and the 8th week after surgery,samples were taken,photographed,and the joints were roughly scored to observe the repair.After paraformaldehyde fixation,decalcification,paraffin embedding,sectioning,HE staining,reddish green Staining and immunohistochemical staining and histological scoring were used to observe the repair between the separate groups.Results:The results of the first part:Fourier transform infrared spectroscopy and nuclear magnetic resonance spectroscopy showed that the pullulan was successfully modified with anhydride to make it UV-curable.The Young's modulus test results show that the Young's modulus of 4%hydrogel is 0.025±0.006 MPa,8%is 0.069 ±0.003 MPa,12%is 0.103± 0.003 MPa,and 16%is 0.175±0.013 MPa.The greater the concentration of the hydrogel,the greater the Young's modulus.The swelling ratio test results showed that the swelling rate of 6%hydrogel was about 50.53%,the swelling rate of 8%hydrogel was 42.38%,and the swelling rate of 10%hydrogel was 37.88%,14%of water condensation.The swelling ratio of the glue is 29.67%,and the higher the concentration,the lower the swelling rate of the hydrogel.The Young's modulus of the glue,the swelling ratio,etc.,show a definite linear correlation with the increase of the hydrogel concentration.The results of scanning electron microscopy showed that pullulan had good through-hole characteristics,the pore size range of the 8%pullulan hydrogel is 121.27±47.58pm,and its network pores were similar to the network structure of collagen,sodium alginate and other hydrogels,and the pore size formed was relatively uniform.The hydrogel can be modified to have a good pore size and a suitable Young's modulus,and is structurally similar to the structure of the extra-cartilage matrix.The results of the second part:The results of calcein/PI staining showed that with the increase of culture time,the number of cells increased,the cells in each hydrogel group proliferated well,and the survival rate was high.The results of HE staining showed that more BBC group and BTP group were observed.Cartilage lacuna,Safranin O staining,GAG/DNA detection,immunofluorescence staining of COL2al showed chondrocyte-specific extracellular matrix(glycosaminoglycan and type ? collagen)in the pullulan group and the collagen hydrogel group The secretion of protein was similar;RT-PCR(COL2a1,SOX9,ACAN,COL1a1,COL10a1)showed that the expression of cartilage-specific marker genes COL2a1,SOX9,ACAN and collagen group in the pullulan hydrogel group Similar,even higher.The results of the third part:The results of in vivo experiments showed that the effect of repair of cartilage defects in the BP group was similar to that of the BC group.Neonatal cartilage tissue was similar to adjacent normal cartilage tissue,and the effect was significantly better than that of group B and BS.The results of histological staining were also consistent with the general observations.Photo-cured modified pullulan hydrogel composite stem cells were used to repair and regenerate cartilage defects in vivo.Conclusion:The anhydride-modified pullulan can be UV-cured into a gel with good through-hole properties,swelling ratio and mechanical strength;the photocopiable modified pullulan hydrogel has the same good in vitro as collagen hydrogel.Inducing bone marrow mesenchyme stem cells to differentiate into cartilage;photocopiable modified pullulan hydrogel composite stem cells have good regenerative repair effect on cartilage defects in vivo. |
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