The Fabrication Of 3D Printed Composite Gradient Hydrogel Scaffold And Its Effect On Cartilage Repair

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Fabricaton of 3D printing composite gradient hydrogelscaffold and its related properties test Objective: To explore the possibility of fabrication of composite gradient hydrogel scaffolds using 3D printing technology with gelatin and hydroxyapatite(HAP)to simulate the morphology and structure of normal cartilage,and to investigate the characteristics of its structure?mechanical properties and biocompatibility by related tests.Methods:(1)Photosensitive gelatine methacrylamide(GelMA)was synthesized by chemical reaction using type A gelatin and methacrylic anhydride(MA).(2)Four hydrogel scaffolds(15%(w/v)GelMA,15%GelMA/3%HAP,30% GelMA,and 30% GelMA/3%HAP)were prepared under UV light exposure.(3)The properties of swelling,mechanical,degradation and cell compatibility of four hydrogel scaffolds were tested and compared to select a scheme of material concentration ratio which is most suitable for gradient scaffold.(4)The 3D printing technology was used to fabricate the composite gradient hydrogel scaffold(upper layer: 15%GelMA,middle layer: 15%GelMA/3%HAP,lower layer: 30% GelMA/3%HAP)and 4 other kinds of non-gradient scaffolds.The mechanical properties of the 5 kinds of scaffolds were tested.(5)Scanning electron microscope(SEM)was used to observe the pore size of the gradient scaffold.The cell compatibility of the scaffold was measured by observing the cell(BMSCs)adhesion on the scaffold surface.(6)The chondrogenic differentiation of BMSCs in 3D printed composite gradient scaffolds was detected by qRT-PCR and was compared with that in non 3D printed group.Results:(1)When the concentration of GelMA hydrogel increased from 15% to 30%,the swelling rate decreased,and the difference was statistically significant(P < 0.05);Compared with 15% GelMA hydrogels,the mixed hydrogel added with 3%HAP had a lower swelling rate(P < 0.05).(2)The Young's modulus of hydrogel increased when the GelMA concentration increased from 15% to 30%(P < 0.05);There was no significant change in Young's modulus of 15%GelMA afer mixed with 3%HAP(P > 0.05);The Young's modulus of 30%GelMA decreased after mixed with 3%HAP(P < 0.05).(3)The degradation rate of 30%GelMA was lower than that of 15%GelMA;The degradation rate of GelMA hydrogel with the same concentration decreased after mixed with HAP(P < 0.05).(4)The cell activity in 15% GelMA hydrogels was better than that of 30%GelMA hydrogel after 72h(P < 0.05);The mixture of 3%HAP with the same concentration of GelMA had no significant effect on the cell activity in hydrogel(P >0.05).(5)The pore size of the 3D printing composite gradient hydrogel scaffold was 397.2 + 15.6?m;The scaffold had good cellular compatibility and Bone mesenchymal stem cells(BMSCs)could adhere on its surface and grew well.(6)The compression modulus test showed that the anti compression performance of the composite gradient hydrogel scaffold was better than those of non-gradient hydrogel scaffold with 15% GelMA and 15% GelMA/3%HAP(P < 0.05).(7)The results of qRT-PCR showed that BMSCs had better chondrogenic differentiation ability in 3D printed composite gradient scaffold group than that in non 3D printed group(P < 0.05).Conclusion: The GelMA hydrogel with low concentration has good biocompatibility,but the biocompatibility of GelMA hydrogel decreases with the increase of concentration;The swelling and mechanical strength of 30% GelMA hydrogel scaffold are better than those with low concentration(15%);The mixture of low concentration of HAP with GelMAhas no affect on its biocompatibility;The composite gradient hydrogel scaffold fabricated by 3D printing can simulate the morphological characteristics of normal cartilage,and has good biocompatibility and mechanical properties,which is superior to the traditional pure GelMA non-gradient hydrogel scaffolds;It also promoted the differentiation of BMSCs into cartilage.Section ? Study on the effect of 3D printing composite gradienthydrogel scaffold on the repair of cartilage injuries Objective: To discuss the effect of 3D printing composite gradient hydrogel scaffold on the repair of cartilage injuries without loading of cells by animal experiment.Methods: Two different hydrogel scaffolds were prepared by 3D printing method(group A: upper layer: 15%GelMA,middle layer: 15%GelMA/3%HAP,lower layer: 30% GelMA/3%HAP gradient hydrogel scaffold;group B: 15% GelMA non-gradient hydrogel scaffold).18 New Zealand white rabbits were randomly divided into 3 groups.Full-thickness cartilage defects(3.5mm in diameter,3mm in depth)were made in the center of femoral trochlea in both knees of each rabbit.The gradient scaffolds(group A)and the non-gradient scaffolds(group B)were implanted separately into the cartilage defects in two groups,and the group C was used as a blank control.3 rabbits in each group were sacrificed in each group by the end of the fourth and twelfth week after operation and their knees were harvested.The outcome was assessed and compared macroscopically by using theInternational Cartilage Repair Society(ICRS)evaluation system first.Then the HE?Safranin O?Collagen II?Green-SafraninO staining and the O'Driscoll histologic scoring system were used to evaluate and compare the repair effect of osteochondral injuries between 3 groups.Results: Compared with group C andgroup B,the 3D printing composite gradient hydrogel scaffolds(group A)had better repair effect in full-thickness cartilage defects,and the repaired tissues were closely connected with the host tissues,the morphology was more similar with the normal cartilage,and the calcified cartilage layer could be observed in some sections in group A,although there were still a small amount of depressions on the surface.The score of ICRS and O 'Driscoll in group A were both higher than those in group B and group C at 4 and 12 weeks(P<0.05).The score of ICRS and O 'Driscoll in group B were both higher than those in group C at 12 weeks(P<0.05).Conclusion: The 3D printing composite gradient scaffold showed good biocompatibility and biodegradability in experimental animals.The scaffold can be implanted in situ by press-fit method,and the experimental results confirmed that the scaffold can be closely fitted within the cartilage defect.The 3D printing composite gradient hydrogel scaffold has good effect on cartilage repair and subchondral bone formation even without loading cells.