甲基丙烯酸酐化明胶-甲壳素纳米晶须生物墨水的特性和3D打印效果

Research on the characteristics and printing effect of chitin nanocrystal-gelatin methacrylate new bioink

目的探讨由甲基丙烯酸酐化明胶(GelMA)和甲壳素纳米晶须(ChiNC)组成的混合生物墨水的物理特征、生物相容性,以及3D打印效果。方法2021年5月至2022年12月,于100mg/mlGelMA生物墨水中添加ChiNC,制备成ChiNC浓度分别为5、10、20mg/ml的混合生物墨水,分别命名为GC5、GC1O、GC20组,与未添加ChiNC的GelMA生物墨水(CCO组)共同实验。采用扫描电镜观察4组生物墨水光固化后形成的水凝胶的横截面,计算孔隙率。称量各组水凝胶溶胀前后质量,计算平衡溶胀比。将4组生物墨水加入48孔板中,光固化成水凝胶,表面种植入脐静脉内皮细胞(HUVECs),于培养基中培养至第1、3、7天分别行CCK-8实验检测吸光度A值,比较4组水凝胶中细胞的增殖速度。4组生物墨水中添加HUVECs,打印网格状支架,于培养基中培养至第1、7天分别行Live-Dead染色,观察细胞存活率。采用打印挤出成丝实验,观察各组生物墨水挤出时的连续成丝性,比较各组的打印效果。采用最佳配比的混合生物墨水3D打印模拟膀胱壁黏膜层、黏膜下层和肌层解剖结构的组织工程膀胱补片,观察打印结构的稳定性和保真度,验证混合生物墨水打印多层复杂结构的可行性。结果扫描电镜观察结果显示,GCO、GC5、GC1O、GC20组水凝胶的孔隙率分别为(51.43±6.23)%、(51.85±6.47)%、(50.55±4.59)%和(42.49±2.20)%,GC0组与其他3组比较差异均无统计学意义(P=0.9994、P=0.9948、P=0.1200)。GC0组平衡溶胀比为9.37±0.49,低于GC5组(8.81±0.41,P=0.0457)、GC10组(7.95±0.19,P<0.01)、GC20组(7.71±0.14,P<0.01),差异均有统计学意义。CCK-8检测结果显示,GCO、GC5、GC10、GC20组培养第1天的吸光度A值分别为0.357±0.0070.350±0.012、0.360±0.009、0.345±0.018,GC10组与其他3组比较差异均无统计学意义(P=0.9332、P=0.5464、P=0.4937);培养第3天的吸光度A值分别为0.634±0.010、0.704±0.009、0.755±0.012、0.653±0.015,GC10组与其他3组比较差异均有统计学意义(P<0.01、P=0.0033、P=0.0002);培养第7天的吸光度A值分别为0.846±0.026、0.930±0.043、1.001±0.031.0.841±0.024,GC10组与其他3组比较差异均有统计学意义(P=0.0012、P=0.1390、P=0.0010)。GCO、CC5、GC10、CC20组培养第1天的HUVECs细胞存活率分别为(90.13±1.63)%、(90.6±2.45)%、(92.58±2.15)%、(91.40±3.17)%,GC0组与其他3组比较差异均无统计学意义(P=0.9869、P=0.3093、P=0.8008);培养第7天的细胞存活率分别为(89.97±3.10)%、(92.18±2.21)%、(92.05±2.25)%、(90.12±1.97)%GC0组与其他3组比较差异均无统计学意义(P=0.3965、P=0.4511、P=0.9995)。打印挤出成丝测试结果显示,GC0组在24℃~25℃时挤出连续且能成丝,GC10组在24℃~27℃时挤出连续且能成丝。使用CC10组打印组织工程膀胱补片,结果显示打印的补片稳定,无塌,保真度高。结论在GelMA中添加ChiNC能促进细胞黏附、增殖,扩大GelMA生物墨水的打印窗口。在GelMA中添加10mg/ml ChiNC制备的混合生物墨水的生物相容性良好,能打印模拟天然膀胱壁解剖结构的组织工程膀胱补片。

Objective This study aimed to investigate the physical properties,biocompatibility,and 3D printing performance of a novel hybrid bioink composed of gelatin methacrylated(GelMA)and chitin nanocrystal(ChiNC).Methods The study was conducted from May 2021 to December 2022,four different bioinks were prepared by adding varying amounts of ChiNC to GelMA bioink.The GelMA concentration in all four bioinks was 100 mg/ml,while the ChiNC concentrations were O mg/ml(no ChiNC added),5 mg/ml,10 mg/ml,and 20 mg/ml,respectively,named as GCO,GC5,GC10,and GC20 bioinks.The crosssectional morphology of the hydrogels formed after photocuring the four bioinks was observed using scanning electron microscopy,and the porosity was calculated.Weighing the hydrogels before and after swelling,and then calculate the equilibrium swelling rate.HUVECs were seeded on the surfaces of the hydrogels prepared from the four bioinks and cultured in medium.Cell proliferation was assessed using CCK-8 assays at ld,3d,and 7d to compare the proliferation rates of cells on the four hydrogels.HUVECs were added to the four bioinks,and grid-like scaffolds were printed and cultured in medium.Live-Dead staining was performed at 1d and 7d to observe cell viability.Compare the printing effect of bioinks by observing its forming continuous threads properties during extrusion.Finally,tissue-engineered bladder patches simulating the mucosal layer,submucosal layer,and muscular layer anatomical structures of the bladder wall were 3D bioprinted using the optimized bioink composition,and the stability and fidelity of the printed structures were observed to further validate the feasibility of printing multi-layered complex structures with the bioink.Results Scanning electron microscopy revealed that the porosity of the GCO,GC5,GC10,and GC20 hydrogels were(51.43±6.23)%,(51.85±6.47)%,(50.55±4.59)%,and(42.49±2.20)%,respectively.The differences in porosity between the GCO group and the other three groups were not statistically significant(P=0.9994,P=0.9948,P=0.1200).The equilibrium swelling ratio of the other three groups[(8.81±0.41),(7.95±0.19),(7.71±0.14)]was significantiy lower than that of the GC0 group(9.37±0.49),and the differences were statistically significant(P=0.0457,P<0.01,P<0.01).CCK-8 assay showed no significant difference in absorbance value between the GC10 group(0.360±0.009)and the CCO group(0.357±0.007),GC5 group(0.350±0.012),and GC20 group(0.345±0.018)on the first day(P=0.9332,P=0.5464,P=0.4937).However,on the third day,the absorbance value of the CC10 group(0.755±0.012)was significantly higher than that of the CCO group(0.634±0.010),GC5 group(0.704±0.009),and GC20 group(0.653±0.015)(P<0.01,P=0.0033,P=0.0002).On the seventh day,the absorbance value of the GC10 group(1.001±0.031)was significantly higher than that of the GCO group(0.846±0.026),GC5 group(0.930±0.043),and CC20 group(0.841±0.024)(P=0.0012,P=0.1390,P=0.0010).The addition of human umbilical vein endothelial cells(HUVECs)into the four groups of hydrogels enabled the printing of grid-like scaffolds,and Live-Dead staining was performed on day 1 and day 7.The cell viability of HUVECs in the four groups on day 1 was(90.13±1.63)%,(90.6±2.45)%,(92.58±2.15)%,and(91.40±3.17)%,respectively.There were no statistically significant differences between the GC0 group and the other three groups(P=0.9869,P=0.3093,P=0.8008).On day 7,the cell viability was(89.97±3.10)%,(92.18±2.21)%,(92.05±2.25)%,and(90.12±1.97)%for the four groups,respectively.There were no statistically significant differences between the GCO group and the other three groups(P=0.3965,P=0.4511,P=0.9995).Bioink extrusion test showed that the GCo hydrogel could be extruded continuously and form threads at temperatures between 24℃and 25℃,while the GC10 hydrogel could be extruded continuously and form threads at temperatures between 24℃and 27℃.Printing tissue engineered bladder patches simulating the anatomical structure of the bladder mucosal layer,submucosal layer,and muscular layer using GC1o bioink,and the printed patches were stable,without collapse,and had high fidelity.Conclusions Adding ChiNC to GelMA promotes cell adhesion,proliferation,and expands the printing window of GelMA bioink.The biocompatibility of the mixed bioink prepared by adding 10 mg/ml ChiNC in GelMA is good,capable of printing tissue-engineered bladder patches that mimic the anatomical structure of natural bladder walls.