改性纳米生物玻璃水凝胶的制备及特性初步研究

Preparation and preliminary research on the characteristics of modified nano-bioglass hydrogel

目的初步探讨改性纳米生物玻璃水凝胶的制备以及它的理化、生物学特性。方法(1)取400 mL氢氧化钙饱和溶液,加入纳米二氧化硅悬液67 mL,制备纳米生物玻璃悬液,观察其悬浮稳定性。(2)制备终质量分数为10%明胶、1%海藻酸钠的水凝胶,设为对照组;在对照组水凝胶基础上加入实验(1)制备的纳米生物玻璃悬液,制备终质量分数为0.5%生物玻璃、10%明胶、1%海藻酸钠的水凝胶,设为实验组。观察2组水凝胶在4、25℃的成胶情况并记录成胶时间及在37℃的熔解情况并记录熔胶时间。另取2组水凝胶,4℃冷浴后用25 g/L的氯化钙溶液交联,用杨氏模量测定仪测量压缩模量。另取2组水凝胶,同前交联后于-20℃冻干,测量相关体积并计算孔隙率。样本数均为3。(3)取12只24 h龄C57BL/6J小鼠乳鼠,分离培养成纤维细胞(Fb),倒置显微镜下观察其形态,并培养第3代Fb,用于后续实验。取Fb,制备细胞浓度为1×10^5个/mL的单细胞悬液,按随机数字表法(下同)分为实验组和对照组,分别加入实验(2)制备的实验组和对照组液态水凝胶,培养12、24、48 h,每组各取3孔,采用细胞计数试剂盒8法检测细胞存活率。(4)取第3代Fb,制备细胞浓度为(3.0~4.5)×10^7个/mL的单细胞悬液,分为实验组和对照组,每组1管,加入绿色荧光探针DIO染色,分别加入实验(2)中制备的实验组和对照组液态水凝胶9 mL,同前交联后制备载细胞水凝胶。培养3 d,激光共聚焦显微镜下观察细胞在凝胶中的存活情况;同前制备载细胞水凝胶块但不加绿色荧光探针DIO,培养7 d,在扫描电子显微镜下观察细胞在水凝胶中的黏附及伸展情况。(5)取6周龄雌性BALB/c-nu裸鼠12只,分为实验组和对照组,每组6只,在背部制作直径为1 cm的圆形全层皮肤缺损创面,伤后即刻,分别放入1块实验(4)制备的实验组和对照组载细胞水凝胶块。伤后7、14 d,每组取3只裸鼠,收集创面及创周组织,苏木精-伊红染色,观察创面愈合情况。对数据行独立样本t检验。结果(1)纳米生物玻璃粒子可在水中均匀分散,具有良好的悬浮稳定性。(2)2组水凝胶在37℃下均呈熔融状态,均未见粒子析出。实验组和对照组水凝胶在37℃下的熔胶时间分别为5、10 min,在25℃下成胶时间分别为30、180 min,在4℃下成胶时间分别为5、10 min。实验组水凝胶压缩模量为(53±6)kPa,明显高于对照组的(23±6)kPa(t=6.364,P<0.01)。实验组水凝胶孔隙率为(86.1±2.1)%,与对照组的(88.2±4.4)%相近(t=1.210,P>0.05)。(3)细胞呈长梭形,细胞核所占比例较大,符合Fb形态学特征。培养12、24、48 h,实验组细胞存活率为(84±4)%、(89±4)%、(130±10)%,与对照组的(89±5)%、(90±4)%和(130±11)%相近(t=1.534、0.611、0.148,P>0.05)。(4)培养3 d,2组细胞在水凝胶中形态完整,未见细胞核裂解、消失,细胞质保持完好,并且实验组细胞荧光强度明显强于对照组。培养7 d,实验组和对照组细胞在水凝胶中黏附、伸展,且实验组细胞在水凝胶中黏附数明显多于对照组。(5)伤后7 d,对照组、实验组裸鼠创面面积均缩小,且实验组减小更明显,2组裸鼠创面及创周均可见大量炎症细胞分布。伤后14 d,对照组裸鼠创面面积大于实验组,且创面及创周炎症细胞明显多于实验组。结论纳米生物玻璃水凝胶具有良好的理化、生物学特性和载细胞潜能,同时还具有促创面愈合能力,在临床应用方面有着较好的潜力。

Objective To explore the preparation and preliminary research on the characteristics of modified nano-bioglass hydrogel.Methods(1)The nano-bioglass suspension was prepared by adding 67 mL nano-silica suspension into 400 mL saturated calcium hydroxide solution,and its suspension stability was observed.(2)The hydrogel with final mass fraction of 10%gelatin and 1%sodium alginate was prepared and set as control group.On the basis of the hydrogel in control group,the nano-bioglass suspension prepared in experiment(1)was added to prepare the hydrogel with the final mass fraction of 0.5%bioglass,10%gelatin,and 1%sodium alginate,and the hydrogel was set as the experimental group.The gelling time at 4 and 25℃and the dissolution time at 37℃of hydrogel in 2 groups were recorded,and the gelation at 4 and 25℃and dissolution condition at 37℃of the hydrogel in 2 groups were observed.The hydrogel in 2 groups were collected and cross-linked with 25 g/L calcium chloride solution after cold bath at 4℃,and the compression modulus was measured by Young′s modulus tester.In addition,the hydrogel in 2 groups were collected and cross-linked as before,and freeze-drying hydrogel was made at -20℃.The relative volumes were measured and the porosity of hydrogel in 2 groups was calculated.The number of sample in the experiment was 3.(3)Fibroblasts(Fbs)were isolated and cultured from 12 C57BL/6J mice of 24 hours old and the morphology was observed by inverted microscope,and the third passage of Fbs were cultured for the following experiment.Fbs were collected to make single cell suspension with the cell concentration of 1×10^5/mL.The single cell suspension was divided into experimental group and control group according the random number table(the same grouping method below),which were added with hydrogel in experimental group and control group prepared in experiment(2),respectively.At culture hour 12,24,and 48,cells of 3 wells in each group were collected to detect the survival rate by cell counting kit 8 method.(4)The third passage Fbs were collected to prepare the single cell suspension with the cell concentration of(3.0~4.5)×10^7/mL,which was divided into experimental group and control group,with 1 tube in each group.The single cell suspension in 2 groups were added with green fluorescent probe DIO for staining and then added with 9 mL hydrogel in experimental group and control group prepared in experiment(2),respectively.The mixed solution of Fbs and hydrogel in 2 groups was cross-linked as before to make cell-loaded hydrogel.On culture day 3,the survival of cells in the hydrogel was observed by laser confocal microscope.The cell-loaded hydrogel was prepared as before and without added with green fluorescent probe DIO.On culture day 7,the adhesion and extension of cells in the hydrogel were observed by scanning electron microscope.(5)Twelve 6-week-old female BALB/c-nu nude mice were collected and divided into experimental group and control group,with 6 mice in each group.A round full-thickness skin defect wound with diameter of 1 cm was made on the back of each mouse.Immediately after injury,one cell-loaded hydrogel block in the experimental group and the control group prepared in experiment(4)was placed in the wound of each mouse in the experimental group and the control group,respectively.On post injury day(PID)7 and 14,3 nude mice in each group were sacrificed to collect the wound and wound margin tissue,which was stained with hematoxylin-eosin to observe the wound healing.Data were statistically analyzed with independent sample t test.Results(1)The nano-bioglass particles could be uniformly dispersed in water and had good suspension stability.(2)The hydrogels of the 2 groups were molten at 37℃,and no precipitation of particle was observed.The dissolving time of the hydrogel in the experimental group and the control group at 37℃ was 5 and 10 min,respectively.The gelation time of the hydrogel in the experimental group and the control group at 25℃ was 30 and 180 min,respectively,and the gelation time of the 2 groups at 4℃ was 5 and 10 min,respectively.The compression modulus of hydrogel in the experimental group was(53±6)kPa,which was significantly higher than(23±6)kPa in the control group(t=6.364,P<0.01).The porosity of the hydrogel in the experimental group was(86.1±2.1)%,which was similar to(88.2±4.4)%in the control group(t=1.210,P>0.05).(3)The cells were in long fusiform,and the proportion of nuclei was high,which was accorded with the morphological characteristics of Fbs.At culture hour 12,24,and 48,the survival rate of cells in the experimental group was(84±4)%,(89±4)%,and(130±10)%,which was similar to(89±5)%,(90±4)%,and(130±11)%in the control group,respectively(t=1.534,0.611,0.148,P>0.05).(4)On culture day 3,the cells in the two groups had complete morphology in the hydrogel,no nuclear lysis or disappearance were observed,the cytoplasm remained intact,and the fluorescence intensity of the cells in the experimental group was significantly stronger than that in the control group.On culture day 7,the cells in the experimental group and the control group adhered and stretched in the hydrogel,and the number of cells in the experimental group adhered to the hydrogel was significantly more than that in the control group.On PID 7,the wound area of the nude mice in the control group and the experimental group were reduced,the reduction area of mice in the experimental group was more obvious,and a large amount of inflammatory cells were seen in and around the wound in the 2 groups.On PID 14,the wound area of the nude mice in the control group was larger than that of the experimental group,and the number of inflammatory cells in and around the wound was significantly more than that in the experimental group.Conclusions Nano-bioglass hydrogel possesses good physical,chemical,and biological properties,cell loading potential,and the ability to promote wound healing,which means it has a good potential in clinical application.