Effect Of Photocuring 3D-Printed Guar Gum Scaffold On Repairing Cartilage Defect

Objective:This project aimed to study the effect of natural plant derived polysaccharide polymer guar gum(GG)on the proliferation and chondrogenic differentiation of bone marrow mesenchymal stem cells and the repair of cartilage defects in vivo,using 3D bio-printing technology.Methods:The method of part one:Photocurable chemically modification and characterization of GG:GG powder was dissolved in distilled water,heated in a water bath,and stirred constantly.After GG completely dissolved,methacrylic anhydride(MAA)was added for chemical modification.The final reaction products were dialyzed,purified,and freeze-dried to obtain guar gum methacrylate(GG-MA1,GG-MA2)with high and low graft rates of methacryloyl(MA).Compared with the raw GG,the chemical composition of the GG-MA was analyzed by FTIR and H~1NMR to verify the success of the modification.Several groups of GG-MA hydrogel inks with different concentrations were prepared,and3D printer was used to screen the appropriate printing concentrations of GG-MA hydrogel inks with good printing effect,high resolution of scaffold structure and relatively low air pressure.Photocuring 3D printed GG-MA hydrogel scaffolds were characterized by scanning electron microscopy(SEM)to observe the interior morphology and pore structure and measuring the aperture size.And a mechanical tester was used to measure the compressive modulus of GG-MA hydrogel scaffold.The degradation rate and the swelling rate of GG-MA hydrogel scaffold were measured in different time points in PBS buffer,simulating in vivo environment.The method of the second part:Induction of cartilage differentiation of bone marrow mesenchyme stem cells(BMSCs)by photocuring 3D printed GG-MA hydrogel scaffolds in vitro:BMSCs were extracted from New Zealand white rabbit and cultured in vitro,and the third generation of stem cells were mixed with sodium alginate hydrogel inks(AS,as the control group)and GG-MA hydrogel inks(experimental group)with high and low modified grafting rates(GG-MA1and GG-MA2)as bio-inks,and then the scaffolds were printed by 3D bio-printers and cultured in cartilage induction medium.Live/Dead staining was used to observe the growth and proliferation of BMSCs in different groups of hydrogel scaffolds under laser confocal microscope at two culture points of 7 and 14 days,respectively.GAG/DNA contents of each group were measured to observe the secretion of cartilage matrix products GAG,glycosaminoglycans.The expression levels of ACAN,COL2a1,COL1a1 and SOX9,the marker genes related to chondrogenic differentiation,were detected by fluorescence quantitative PCR to verify the effect of chondrogenic differentiation of BMSCs in each group.The method of the third part:Study on the effect of photocuring 3D printed GG-MA hydrogel scaffolds on the repair of cartilage defects in vivo:New Zealand white rabbit articular cartilage defect models were established.Different groups of 3D printed hydrogel scaffolds with rabbit BMSCs(AS,GG-MA1,GG-MA2group)were implanted into the defect areas and the defect joints samples were gathered after 12 weeks.To assess the effect of repairment,we took photos for the defect areas and calculated general repair scores.After fixed by paraformaldehyde,decalcification,paraffin embedding,sectioning,the specimens were conducted with HE staining,safranin O-green staining and histological scoring were used to observe the repair between the separate groups.Results:The results of the first part:The results of FTIR and H~1 NMR showed that the methacrylate anhydride modification was successful,and the chemically modified GG-MA hydrogel had the property of UV curing.The appropriate printing concentration of GG-MA hydrogel inks was 4%w/v after screened by 3D printer.Compressive modulus test results showed that the compressive modulus of the GG-MA1 hydrogel scaffold with a concentration of 4%w/v was 0.069 MPa,and that of the GG-MA2 hydrogel scaffold was 0.184 MPa.The swelling rate test results showed that the swelling rate of GG-MA1 hydrogel scaffold was about1917%,and that of GG-MA2 hydrogel scaffold was 1139%.The degradation rate test results in vitro showed that the degradation rate of GG-MA1 hydrogel scaffold was about 90.71%,and that of GG-MA2 hydrogel scaffold was 61.41%.The results of SEM showed the internal pore inter-connecting structure of the photocuring 3D printed guar gum hydrogel scaffolds.The results of the second part:The results of Live/Dead staining showed that with the increase of culture time,the number of cells increased,and the growth and proliferation status of cells in different groups of 3D printed hydrogel scaffolds was good with high survival rate.The results of GAG/DNA test showed that the relative expressions of glycosaminoglycans in AS group and GG-MA2 group increased with time,while the relative expressions of glycosaminoglycans in GG-MA1 group decreased.The results of fluorescence quantitative PCR(ACAN,COL2a1,COL1a1,SOX9)showed that the expression level of cartilage specific marker gene COL2a1 in AS group and GG-MA2 group increased significantly over time,which was even higher in GG-MA2 group.The results of the third part:The gross observation results of the collected joint samples in vivo experiments showed that the repair effect of cartilage defects in the GG-MA2 group was similar to that in the AS group,with the newborn cartilage tissue close to the adjacent normal cartilage tissue,while the repair effect of the GG-MA1 group was poor and mostly fibrous.The histological staining results including HE staining and safranin O-green staining were basically consistent with the general observation results.Conclusion:Natural plant derived polysaccharide GG could be modified by MAA to have the ability to be UV-curable.The UV-curing GG-MA hydrogel scaffolds prepared by 3D bioprinter had a good internal pore inter-connecting structure,good swelling property,degradability,and mechanical strength,which could be adjusted by changing the substitution rate of the modification reaction.The photocuring 3D printed GG-MA hydrogel scaffolds had similar properties to AS hydrogel scaffolds and could provide a good supporting environment for the growth and proliferation as well as chondrogenic differentiation of BMSCs in vitro.Photocuring 3D printed GG-MA hydrogel scaffolds loaded with BMSCs showed a good regeneration and repair effect on cartilage tissue defects in vivo.