Effects of in situ cross-linked graphene oxide-containing gelatin methacrylate anhydride hydrogel on wound vascularization of full-thickness skin defect in mice
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摘要:
目的 制备含氧化石墨烯（GO）的甲基丙烯酸酐化明胶（GelMA）水凝胶并探讨原位光聚合GO-GelMA复合水凝胶对小鼠全层皮肤缺损创面血管化的影响。 方法 采用实验研究方法。将0.2 mg/mL的GO溶液50 μL均匀涂抹于导电胶上，烘干后于场发射扫描电子显微镜下观察GO的结构和大小。将人皮肤成纤维细胞（HSF）分为采用相应终质量浓度GO处理的0 μg/mL GO（不加GO溶液，下同）组、0.1 μg/mL GO组、1.0 μg/mL GO组、5.0 μg/mL GO组、10.0 μg/mL GO组，用酶标仪检测细胞培养48 h的吸光度值，以此表示细胞增殖活性（样本数为6）。将HSF和人脐静脉血管内皮细胞（HUVEC）分别分为采用相应终质量浓度GO处理的0 μg/mL GO组、0.1 μg/mL GO组、1.0 μg/mL GO组、5.0 μg/mL GO组，采用划痕试验检测划痕后24、36 h HSF的迁移率（样本数为5）及划痕后12 h HUVEC的迁移率（样本数为3），采用酶联免疫吸附测定法检测培养4、6、8 h后HSF分泌的血管内皮生长因子（VEGF）水平（样本数为3）。将配制的含相应终质量浓度GO的GO-GelMA复合水凝胶设为0 μg/mL GO复合水凝胶组、0.1 μg/mL GO复合水凝胶组、1.0 μg/mL GO复合水凝胶组、5.0 μg/mL GO复合水凝胶组，观察其交联前后的性状，检测用磷酸盐缓冲液浸泡3、7 d后GO的释放情况（样本数为3）。在16只6周龄雌性C57BL/6小鼠背部制作全层皮肤缺损创面，将采用原位交联的含相应终质量浓度GO的GO-GelMA复合水凝胶处理的小鼠按随机数字表法分为0 μg/mL GO复合水凝胶组、0.1 μg/mL GO复合水凝胶组、1.0 μg/mL GO复合水凝胶组、5.0 μg/mL GO复合水凝胶组，每组4只，观察治疗3、7、14 d创面大体情况并计算创面愈合率，采用激光多普勒血流仪检测治疗3、7、14 d创面血流灌注并计算平均灌注单位（MPU）比值，采用苏木精-伊红染色观察治疗7 d创面血管新生情况并计算血管密度（样本数均为3）。取0 μg/mL GO复合水凝胶组和0.1 μg/mL GO复合水凝胶组治疗7 d的创面组织，采用苏木精-伊红染色观察GO分布与血管新生的关系（样本数为3），行免疫组织化学染色后观察VEGF的表达。对数据行重复测量方差分析、单因素方差分析、Tukey法。 结果 GO为多层片状结构，宽度约为20 μm、长度约为50 μm。培养48 h，10.0 μg/mL GO组HSF的吸光度值明显低于0 μg/mL GO组（q=7.64，P<0.01）。划痕后24 h，4组HSF迁移率相近（P>0.05）；划痕后36 h，0.1 μg/mL GO组HSF迁移率明显高于0 μg/mL GO组、1.0 μg/mL GO组、5.0 μg/mL GO组（q值分别为7.48、10.81、10.20，P值均<0.01）。划痕后12 h，0.1 μg/mL GO组HUVEC迁移率明显高于0 μg/mL GO组、1.0 μg/mL GO组、5.0 μg/mL GO组（q值分别为7.11、8.99、14.92，P值均<0.01），5.0 μg/mL GO组HUVEC迁移率明显低于0 μg/mL GO组和1.0 μg/mL GO组（q值分别为7.81、5.33，P<0.05或P<0.01）。培养4、6 h，4组HSF的VEGF表达均相近（P>0.05）；培养8 h，0.1 μg/mL GO组HSF的VEGF表达明显高于0 μg/mL GO组和5.0 μg/mL GO组（q值分别为4.75、4.48，P值均<0.05）。4组GO-GelMA复合水凝胶在交联前均呈红色液体状，交联后呈微黄色凝胶状且流动性无明显差异。0 μg/mL GO复合水凝胶组复合水凝胶各时间点均无GO释放，其余3组GO-GelMA复合水凝胶中的GO于浸泡3 d部分释放，至浸泡7 d全部释放。治疗3~14 d，4组小鼠创面可见水凝胶敷料覆盖在位并保持湿润，创面逐渐愈合。治疗3、7、14 d，4组小鼠创面愈合率均相近（P>0.05）。治疗3 d，0.1 μg/mL GO复合水凝胶组小鼠创面MPU比值明显高于0 μg/mL GO复合水凝胶组、1.0 μg/mL GO复合水凝胶组、5.0 μg/mL GO复合水凝胶组（q值分别为10.70、11.83、10.65，P<0.05或P<0.01）。治疗7、14 d，4组小鼠创面MPU比值均相近（P>0.05）。0.1 μg/mL GO复合水凝胶组小鼠创面治疗7 d的MPU比值明显低于治疗3 d（q=14.38，P<0.05），治疗14 d的MPU比值明显低于治疗7 d（q=27.78，P<0.01）。治疗7 d，0.1 μg/mL GO复合水凝胶组小鼠创面新生血管密度为每200倍视野下（120.7±4.1）根，明显高于0 μg/mL GO复合水凝胶组、1.0 μg/mL GO复合水凝胶组、5.0 μg/mL GO复合水凝胶组的每200倍视野下（61.7±1.3）、（77.7±10.2）、（99.0±7.9）根（q值分别为12.88、7.79、6.70，P值均<0.01）；1.0 μg/mL GO复合水凝胶组和5.0 μg/mL GO复合水凝胶组小鼠创面新生血管密度均明显高于0 μg/mL GO复合水凝胶组（q值分别为5.10、6.19，P<0.05）。治疗7 d，相较于0 μg/mL GO复合水凝胶组，0.1 μg/mL GO复合水凝胶组小鼠创面中成簇新生血管更多，且聚集于GO附近；0.1 μg/mL GO复合水凝胶组小鼠创面中GO和新生血管分布区域有大量VEGF表达。 结论 GO质量浓度低于10.0 μg/mL对HSF增殖活性无明显影响，0.1 μg/mL的GO能够促进HSF和HUVEC迁移，能促进HSF分泌VEGF。原位光聚合GO-GelMA复合水凝胶敷料能够通过促进小鼠全层皮肤缺损创面血管新生，增加创面早期血流灌注，且GO对新生血管有富集作用，其机制可能与GO促进创面细胞分泌VEGF相关。 Abstract:Objective To prepare graphene oxide (GO)-containing gelatin methacrylate anhydride (GelMA) hydrogel and to investigate the effects of in situ photopolymerized GO-GelMA composite hydrogel in wound vascularization of full-thickness skin defect in mice. Methods The experimental study method was used. The 50 μL of 0.2 mg/mL GO solution was evenly applied onto the conductive gel, and the structure and size of GO were observed under field emission scanning electron microscope after drying. Human skin fibroblasts (HSFs) were divided into 0 μg/mL GO (without GO solution, the same as below) group, 0.1 μg/mL GO group, 1.0 μg/mL GO group, 5.0 μg/mL GO group, and 10.0 μg/mL GO group treated with GO of the corresponding final mass concentration, and the absorbance value was detected using a microplate analyzer after 48 h of culture to reflect the proliferation activity of cells (n=6). HSFs and human umbilical vein vascular endothelial cells (HUVECs) were divided into 0 μg/mL GO group, 0.1 μg/mL GO group, 1.0 μg/mL GO group, and 5.0 μg/mL GO group treated with GO of the corresponding final mass concentration, and the migration rates of HSFs at 24 and 36 h after scratching (n=5) and HUVECs at 12 h after scratching (n=3) were detected by scratch test, and the level of vascular endothelial growth factor (VEGF) secreted by HSFs after 4, 6, and 8 h of culture was detected by enzyme-linked immunosorbent assay method (n=3). The prepared GO-GelMA composite hydrogels containing GO of the corresponding final mass concentration were set as 0 μg/mL GO composite hydrogel group, 0.1 μg/mL GO composite hydrogel group, 1.0 μg/mL GO composite hydrogel group, and 5.0 μg/mL GO composite hydrogel group to observe their properties before and after cross-linking, and to detect the release of GO after soaking with phosphate buffer solution for 3 and 7 d (n=3). The full-thickness skin defect wounds were made on the back of 16 6-week-old female C57BL/6 mice. The mice treated with in situ cross-linked GO-GelMA composite hydrogel containing GO of the corresponding final mass concentration were divided into 0 μg/mL GO composite hydrogel group, 0.1 μg/mL GO composite hydrogel group, 1.0 μg/mL GO composite hydrogel group, and 5.0 μg/mL GO composite hydrogel group according to the random number table, with 4 mice in each group. The general condition of wound was observed and the wound healing rate was calculated on 3, 7, and 14 d of treatment, the wound blood perfusion was detected by laser Doppler flowmetry on 3, 7, and 14 d of treatment and the mean perfusion unit (MPU) ratio was calculated, and the wound vascularization on 7 d of treatment was observed after hematoxylin-eosin staining and the vascular density was calculated (n=3). The wound tissue of mice in 0 μg/mL GO composite hydrogel group and 0.1 μg/mL GO composite hydrogel group on 7 d of treatment was collected to observe the relationship between the distribution of GO and neovascularization by hematoxylin-eosin staining (n=3) and the expression of VEGF by immunohistochemical staining. Data were statistically analyzed with analysis of variance for repeated measurement, one-way analysis of variance, and Tukey's method. Results GO had a multilayered lamellar structure with the width of about 20 μm and the length of about 50 μm. The absorbance value of HSFs in 10.0 μg/mL GO group was significantly lower than that in 0 μg/mL GO group after 48 h of culture (q=7.64, P<0.01). At 24 h after scratching, the migration rates of HSFs were similar in the four groups (P>0.05); at 36 h after scratching, the migration rate of HSFs in 0.1 μg/mL GO group was significantly higher than that in 0 μg/mL GO group, 1.0 μg/mL GO group, and 5.0 μg/mL GO group (with q values of 7.48, 10.81, and 10.20, respectively, P<0.01). At 12 h after scratching, the migration rate of HUVECs in 0.1 μg/mL GO group was significantly higher than that in 0 μg/mL GO group, 1.0 μg/mL GO group, and 5.0 μg/mL GO group (with q values of 7.11, 8.99, and 14.92, respectively, P<0.01), and the migration rate of HUVECs in 5.0 μg/mL GO group was significantly lower than that in 0 μg/mL GO group and 1.0 μg/mL GO group (with q values of 7.81 and 5.33, respectively, P<0.05 or P<0.01 ). At 4 and 6 h of culture, the VEGF expressions of HSFs in the four groups were similar (P>0.05); at 8 h of culture, the VEGF expression of HSFs in 0.1 μg/mL GO group was significantly higher than that in 0 μg/mL GO group and 5.0 μg/mL GO group (with q values of 4.75 and 4.48, respectively, P<0.05). The GO-GelMA composite hydrogels in the four groups were all red liquid before cross-linking, which turned to light yellow gel after cross-linking, with no significant difference in fluidity. The GO in the GO-GelMA composite hydrogel of 0 μg/mL GO composite hydrogel group had no release of GO at all time points; the GO in the GO-GelMA composite hydrogels of the other 3 groups was partially released on 3 d of soaking, and all the GO was released on 7 d of soaking. From 3 to 14 d of treatment, the wounds of mice in the 4 groups were covered with hydrogel dressings, kept moist, and gradually healed. On 3, 7, and 14 d of treatment, the wound healing rates of mice in the four groups were similar (P>0.05). On 3 d of treatment, the MPU ratio of wound of mice in 0.1 μg/mL GO composite hydrogel group was significantly higher than that in 0 μg/mL GO composite hydrogel group, 1.0 μg/mL GO composite hydrogel group, and 5.0 μg/mL GO composite hydrogel group (with q values of 10.70, 11.83, and 10.65, respectively, P<0.05 or P<0.01). On 7 and 14 d of treatment, the MPU ratios of wound of mice in the four groups were similar (P>0.05). The MPU ratio of wound of mice in 0.1 μg/mL GO composite hydrogel group on 7 d of treatment was significantly lower than that on 3 d of treatment (q=14.38, P<0.05), and that on 14 d of treatment was significantly lower than that on 7 d of treatment (q=27.78, P<0.01). On 7 d of treatment, the neovascular density of wound of mice on 7 d of treatment was 120.7±4.1 per 200 times of visual field, which was significantly higher than 61.7±1.3, 77.7±10.2, and 99.0±7.9 per 200 times of visual field in 0 μg/mL GO composite hydrogel group, 1.0 μg/mL GO composite hydrogel group, and 5.0 μg/mL GO composite hydrogel group (with q values of 12.88, 7.79, and 6.70, respectively, P<0.01), and the neovascular density of wound of mice in 1.0 μg/mL GO composite hydrogel group and 5.0 μg/mL GO composite hydrogel group was significantly higher than that in 0 μg/mL GO composite hydrogel group (with q values of 5.10 and 6.19, respectively, P<0.05). On 7 d of treatment, cluster of new blood vessels in wound of mice in 0.1 μg/mL GO composite hydrogel group was significantly more than that in 0 μg/mL GO composite hydrogel group, and the new blood vessels were clustered near the GO; a large amount of VEGF was expressed in wound of mice in 0.1 μg/mL GO composite hydrogel group in the distribution area of GO and new blood vessels. Conclusions GO with mass concentration lower than 10.0 μg/mL had no adverse effect on proliferation activity of HSFs, and GO of 0.1 μg/mL can promote the migration of HSFs and HUVECs, and can promote the secretion of VEGF in HSFs. In situ photopolymerized of GO-GelMA composite hydrogel dressing can promote the wound neovascularization of full-thickness skin defect in mice and increase wound blood perfusion in the early stage, with GO showing an enrichment effect on angiogenesis, and the mechanism may be related to the role of GO in promoting the secretion of VEGF by wound cells. -
参考文献
(43) [1] GuillemotF,SouquetA,CatrosS,et al.High-throughput laser printing of cells and biomaterials for tissue engineering[J].Acta Biomater,2010,6(7):2494-2500.DOI: 10.1016/j.actbio.2009.09.029. [2] CampbellPG,WeissLE.Tissue engineering with the aid of inkjet printers[J].Expert Opin Biol Ther,2007,7(8):1123-1127.DOI: 10.1517/14712598.7.8.1123. [3] OzbolatIT.Bioprinting scale-up tissue and organ constructs for transplantation[J].Trends Biotechnol,2015,33(7):395-400.DOI: 10.1016/j.tibtech.2015.04.005. [4] LiX, LianQ, LiD, et al. Development of a robotic arm based hydrogel additive manufacturing system for in-situ printing[J]. Applied Sciences, 2017, 7(1):73. DOI: 10.3390/app7010073. [5] SinghS,ChoudhuryD,YuF,et al.In situ bioprinting - bioprinting from benchside to bedside?[J].Acta Biomater,2020,101:14-25.DOI: 10.1016/j.actbio.2019.08.045. [6] HuangR,HuJ,QianW,et al.Recent advances in nanotherapeutics for the treatment of burn wounds[J/OL].Burns Trauma,2021,9:tkab026[2022-03-14].https://pubmed.ncbi.nlm.nih.gov/34778468/.DOI: 10.1093/burnst/tkab026. [7] WangH,LiuY,CaiK,et al.Antibacterial polysaccharide-based hydrogel dressing containing plant essential oil for burn wound healing[J/OL].Burns Trauma,2021,9:tkab041[2022-03-14].https://pubmed.ncbi.nlm.nih.gov/34988231/.DOI: 10.1093/burnst/tkab041. [8] AfewerkiS,SheikhiA,KannanS,et al.Gelatin-polysaccharide composite scaffolds for 3D cell culture and tissue engineering: towards natural therapeutics[J].Bioeng Transl Med,2018,4(1):96-115.DOI: 10.1002/btm2.10124. [9] LiMN,YuHP,KeQF,et al.Gelatin methacryloyl hydrogels functionalized with endothelin-1 for angiogenesis and full- thickness wound healing[J].J Mater Chem B,2021,9(23):4700-4709.DOI: 10.1039/d1tb00449b. [10] VandoorenJ,Van den SteenPE,OpdenakkerG.Biochemistry and molecular biology of gelatinase B or matrix metalloproteinase-9 (MMP-9): the next decade[J].Crit Rev Biochem Mol Biol,2013,48(3):222-272.DOI: 10.3109/10409238.2013.770819. [11] JangMJ,BaeSK,JungYS,et al.Enhanced wound healing using a 3D printed VEGF-mimicking peptide incorporated hydrogel patch in a pig model[J].Biomed Mater,2021,16(4):605.DOI: 10.1088/1748-605X/abf1a8. [12] LoessnerD,MeinertC,KaemmererE,et al.Functionalization, preparation and use of cell-laden gelatin methacryloyl-based hydrogels as modular tissue culture platforms[J].Nat Protoc,2016,11(4):727-746.DOI: 10.1038/nprot.2016.037. [13] KlotzBJ,GawlittaD,AJWPRosenberg,et al.Gelatin-methacryloyl hydrogels: towards biofabrication-based tissue repair[J].Trends Biotechnol,2016,34(5):394-407.DOI: 10.1016/j.tibtech.2016.01.002. [14] NuutilaK,SamandariM,EndoY,et al.In vivo printing of growth factor-eluting adhesive scaffolds improves wound healing[J].Bioact Mater,2022,8:296-308.DOI: 10.1016/j.bioactmat.2021.06.030. [15] ZhouX,ChenJ,SunH,et al.Spatiotemporal regulation of angiogenesis/osteogenesis emulating natural bone healing cascade for vascularized bone formation[J].J Nanobiotechnology,2021,19(1):420.DOI: 10.1186/s12951-021-01173-z. [16] ChenYC,LinRZ,QiH,et al.Functional human vascular network generated in photocrosslinkable gelatin methacrylate hydrogels[J].Adv Funct Mater,2012,22(10):2027-2039.DOI: 10.1002/adfm.201101662. [17] LinRZ,ChenYC,Moreno-LunaR,et al.Transdermal regulation of vascular network bioengineering using a photopolymerizable methacrylated gelatin hydrogel[J].Biomaterials,2013,34(28):6785-6796.DOI: 10.1016/j.biomaterials.2013.05.060. [18] FangH,LuoC,LiuS,et al.A biocompatible vascularized graphene oxide (GO)-collagen chamber with osteoinductive and anti- fibrosis effects promotes bone regeneration in vivo[J].Theranostics,2020,10(6):2759-2772.DOI: 10.7150/thno.42006. [19] NorahanMH,AmroonM,GhahremanzadehR,et al.Electroactive graphene oxide-incorporated collagen assisting vascularization for cardiac tissue engineering[J].J Biomed Mater Res A,2019,107(1):204-219.DOI: 10.1002/jbm.a.36555. [20] ChakrabortyS,PonrasuT,ChandelS,et al.Reduced graphene oxide-loaded nanocomposite scaffolds for enhancing angiogenesis in tissue engineering applications[J].R Soc Open Sci,2018,5(5):172017.DOI: 10.1098/rsos.172017. [21] MukherjeeS,SriramP,BaruiAK,et al.Graphene oxides show angiogenic properties[J].Adv Healthc Mater,2015,4(11):1722-1732.DOI: 10.1002/adhm.201500155. [22] ChangTK,LuYC,YehST,et al.In vitro and in vivo biological responses to graphene and graphene oxide: a murine calvarial animal study[J].Int J Nanomedicine,2020,15:647-659.DOI: 10.2147/IJN.S231885. [23] LiuW,LuoH,WeiQ,et al.Electrochemically derived nanographene oxide activates endothelial tip cells and promotes angiogenesis by binding endogenous lysophosphatidic acid[J].Bioact Mater,2022,9:92-104.DOI: 10.1016/j.bioactmat.2021.07.007. [24] 焦德龙 功能化石墨烯/光凝胶复合载药体系用于骨组织再生修复研究 上海 上海交通大学 2019 DOI: 10.27307/d.cnki.gsjtu.2019.004439 焦德龙.功能化石墨烯/光凝胶复合载药体系用于骨组织再生修复研究[D].上海:上海交通大学,2019.DOI:10.27307/d.cnki.gsjtu.2019.004439.
[25] YueK,Trujillo-de SantiagoG,AlvarezMM,et al.Synthesis, properties, and biomedical applications of gelatin methacryloyl (GelMA) hydrogels[J].Biomaterials,2015,73:254-271.DOI: 10.1016/j.biomaterials.2015.08.045. [26] CoentroJQ,PuglieseE,HanleyG,et al.Current and upcoming therapies to modulate skin scarring and fibrosis[J].Adv Drug Deliv Rev,2019,146:37-59.DOI: 10.1016/j.addr.2018.08.009. [27] VedakumariSW,Veda JancySJ,PravinYR,et al.Facile synthesis of sericin modified graphene oxide nanocomposites for treating ischemic diseases[J].Environ Res,2022,209:112925.DOI: 10.1016/j.envres.2022.112925. [28] VerdeV,LongoA,CucciLM,et al.Anti-angiogenic and anti-proliferative graphene oxide nanosheets for tumor cell therapy[J].Int J Mol Sci,2020,21(15):5571.DOI: 10.3390/ijms21155571. [29] LaiPX,ChenCW,WeiSC,et al.Ultrastrong trapping of VEGF by graphene oxide: anti-angiogenesis application[J].Biomaterials,2016,109:12-22.DOI: 10.1016/j.biomaterials.2016.09.005. [30] ZhangY,AliSF,DervishiE,et al.Cytotoxicity effects of graphene and single-wall carbon nanotubes in neural phaeochromocytoma-derived PC12 cells[J].ACS Nano,2010,4(6):3181-3186.DOI: 10.1021/nn1007176. [31] LiangY,ChenB,LiM,et al.Injectable antimicrobial conductive hydrogels for wound disinfection and infectious wound healing[J].Biomacromolecules,2020,21(5):1841-1852.DOI: 10.1021/acs.biomac.9b01732. [32] GurtnerGC,WernerS,BarrandonY,et al.Wound repair and regeneration[J].Nature,2008,453(7193):314-321.DOI: 10.1038/nature07039. [33] LiM,ZhaoY,HaoH,et al.Theoretical and practical aspects of using fetal fibroblasts for skin regeneration[J].Ageing Res Rev,2017,36:32-41.DOI: 10.1016/j.arr.2017.02.005. [34] WangM,WangC,ChenM,et al.Efficient angiogenesis-based diabetic wound healing/skin reconstruction through bioactive antibacterial adhesive ultraviolet shielding nanodressing with exosome release[J].ACS Nano,2019,13(9):10279-10293.DOI: 10.1021/acsnano.9b03656. [35] RidiandriesA,TanJ,BursillCA.The role of chemokines in wound healing[J].Int JTM Mol Sci,2018,19(10):3217.DOI: 10.3390/ijms19103217. [36] JamiesonD,ChanceB,CadenasE,et al.The relation of free radical production to hyperoxia[J].Annu Rev Physiol,1986,48:703-719.DOI: 10.1146/annurev.ph.48.030186.003415. [37] AllenRG,BalinAK.Oxidative influence on development and differentiation: an overview of a free radical theory of development[J].Free Radic Biol Med,1989,6(6):631-661.DOI: 10.1016/0891-5849(89)90071-3. [38] CalabreseV,MancusoC,CalvaniM,et al.Nitric oxide in the central nervous system: neuroprotection versus neurotoxicity[J].Nat Rev Neurosci,2007,8(10):766-775.DOI: 10.1038/nrn2214. [39] SemenzaGL.HIF-1 and mechanisms of hypoxia sensing[J].Curr Opin Cell Biol,2001,13(2):167-171.DOI: 10.1016/s0955-0674(00)00194-0. [40] ChongY,GeC,YangZ,et al.Reduced cytotoxicity of graphene nanosheets mediated by blood-protein coating[J].ACS Nano,2015,9(6):5713-5724.DOI: 10.1021/nn5066606. [41] WeiXQ,HaoLY,ShaoXR,et al.Insight into the interaction of graphene oxide with serum proteins and the impact of the degree of reduction and concentration[J].ACS Appl Mater Interfaces,2015,7(24):13367-13374.DOI: 10.1021/acsami.5b01874. [42] FengR,YuY,ShenC,et al.Impact of graphene oxide on the structure and function of important multiple blood components by a dose-dependent pattern[J].J Biomed Mater Res A,2015,103(6):2006-2014.DOI: 10.1002/jbm.a.35341. [43] LiS,MulloorJJ,WangL,et al.Strong and selective adsorption of lysozyme on graphene oxide[J].ACS Appl Mater Interfaces,2014,6(8):5704-5712.DOI: 10.1021/am500254e. -
2 细胞计数试剂盒8法检测5组人皮肤成纤维细胞培养48 h后的吸光度值 (样本数为6，
注：以吸光度值表示细胞增殖活性；GO为氧化石墨烯；横坐标下方1、2、3、4、5分别为0 μg/mL GO组、0.1 μg/mL GO组、1.0 μg/mL GO组、5.0 μg/mL GO组、10.0 μg/mL GO组；5组细胞吸光度值总体比较，F=17.16，P<0.001；与0 μg/mL GO组比较，aP<0.01
3 划痕试验观察4组人皮肤成纤维细胞划痕后36 h的迁移情况 倒置荧光显微镜×50，图中标尺为500 μm。3A、3B、3C、3D.分别为0 μg/mL GO组、0.1 μg/mL GO组、1.0 μg/mL GO组、5.0 μg/mL GO组细胞迁移情况，图3B剩余划痕面积最小，图3A、3C、3D剩余划痕面积相近且均明显大于图3B
注：GO为氧化石墨烯
4 划痕试验观察4组人脐静脉血管内皮细胞划痕后12 h的迁移情况 倒置荧光显微镜×50，图中标尺为500 μm。4A、4B、4C、4D.分别为0 μg/mL GO组、0.1 μg/mL GO组、1.0 μg/mL GO组、5.0 μg/mL GO组细胞迁移情况，图4B剩余划痕面积最小，图4A、4C、4D剩余划痕面积相似且均大于图4B
注：GO为氧化石墨烯
5 4组GO-GelMA复合水凝胶交联前后性状。5A、5B、5C、5D.分别为交联前0 μg/mL GO复合水凝胶组、0.1 μg/mL GO复合水凝胶组、1.0 μg/mL GO复合水凝胶组、5.0 μg/mL GO复合水凝胶组，均为红色液体状；5E、5F、5G、5H.分别为交联后0 μg/mL GO复合水凝胶组、0.1 μg/mL GO复合水凝胶组、1.0 μg/mL GO复合水凝胶组、5.0 μg/mL GO复合水凝胶组，均为微黄色凝胶状，且5.0 μg/mL GO复合水凝胶组颜色最深，4组流动性无明显差别
注：GO为氧化石墨烯，GelMA为甲基丙烯酸酐化明胶
6 4组小鼠全层皮肤缺损创面经GO-GelMA复合水凝胶治疗各时间点愈合情况。6A、6B、6C、6D.分别为治疗3 d时0 μg/mL GO复合水凝胶组、0.1 μg/mL GO复合水凝胶组、1.0 μg/mL GO复合水凝胶组、5.0 μg/mL GO复合水凝胶组，创面均不同程度缩小；6E、6F、6G、6H.分别为治疗14 d 时0 μg/mL GO复合水凝胶组、0.1 μg/mL GO复合水凝胶组、1.0 μg/mL GO复合水凝胶组、5.0 μg/mL GO复合水凝胶组，创面均基本愈合，且均未见水凝胶敷料残留
注：GO为氧化石墨烯，GelMA为甲基丙烯酸酐化明胶
7 4组小鼠全层皮肤缺损创面经GO-GelMA复合水凝胶治疗各时间点血流灌注情况。7A、7B、7C.分别为治疗3 d时0 μg/mL GO复合水凝胶组、1.0 μg/mL GO复合水凝胶组、5.0 μg/mL GO复合水凝胶组创面血流灌注情况；7D、7E、7F.分别为0.1 μg/mL GO复合水凝胶组治疗3、7、14 d 创面血流灌注情况，随着治疗时间延长，血流灌注逐渐减少，但图7D血流灌注明显高于图7A、7B、7C
注：GO为氧化石墨烯，GelMA为甲基丙烯酸酐化明胶；黑色代表无血流灌注，绿色代表血流灌注少，红色代表血流灌注多
8 4组小鼠全层皮肤缺损创面经GO-GelMA复合水凝胶治疗7 d血管新生情况。8A、8B、8C、8D.分别为0 μg/mL GO复合水凝胶组、0.1 μg/mL GO复合水凝胶组、1.0 μg/mL GO复合水凝胶组、5.0 μg/mL GO复合水凝胶组，均有血管生成 苏木精-伊红×200，图中标尺为100 μm；8E、8F.分别为0 μg/mL GO复合水凝胶组和0.1 μg/mL GO复合水凝胶组，0.1 μg/mL GO复合水凝胶组有成簇新生血管且集中分布于GO附近 苏木精-伊红×400，图中标尺为50 μm
注：GO为氧化石墨烯，GelMA为甲基丙烯酸酐化明胶；黑色箭头指示GO
9 2组小鼠全层皮肤缺损创面经GO-GelMA复合水凝胶治疗7 d时VEGF的表达 二氨基联苯胺-苏木精×400，图中标尺为50 μm。9A、9B.分别为0 μg/mL GO复合水凝胶组、0.1 μg/mL GO复合水凝胶组VEGF表达，0.1 μg/mL GO复合水凝胶组VEGF表达明显高于0 μg/mL GO复合水凝胶组
注：VEGF阳性染色为深褐色；GO为氧化石墨烯，GelMA为甲基丙烯酸酐化明胶，VEGF为血管内皮生长因子；白色箭头指示的是GO
表1 4组人皮肤成纤维细胞划痕后各时间点细胞迁移率比较（%，
组别 样本数 24 h 36 h 0 μg/mL GO组 5 23.5±7.7 55.0±7.2a 0.1 μg/mL GO组 5 30.1±1.1 82.2±13.9 1.0 μg/mL GO组 5 19.1±6.9 42.8±2.6a 5.0 μg/mL GO组 5 19.1±8.9 45.1±9.5a F值 2.85 19.08 P值 0.070 <0.001 注：GO为氧化石墨烯；处理因素主效应，F=1.97，P=0.159；时间因素主效应，F=441.00，P<0.001；两者交互作用，F=0.71，P=0.641；F值、P值为组间各时间点总体比较所得；与0.1 μg/mL GO组比较， aP<0.01 
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表2 4组人皮肤成纤维细胞培养各时间点血管内皮生长因子的表达比较（
组别 样本数 4 h 6 h 8 h 0 μg/mL GO组 3 17.90±0.89 17.14±0.92 17.11±1.28a 0.1 μg/mL GO组 3 17.72±0.45 19.62±1.64 19.56±0.51 1.0 μg/mL GO组 3 16.98±0.53 17.31±1.21 17.76±0.86 5.0 μg/mL GO组 3 16.78±0.59 17.07±0.08 17.25±0.60a F值 2.25 3.61 5.11 P值 0.159 0.065 0.029 注：GO为氧化石墨烯；处理因素主效应，F=8.52，P<0.001；时间因素主效应，F=1.35，P=0.279；两者交互作用，F=1.52，P=0.215；F值、P值为组间各时间点总体比较所得；与0.1 μg/mL GO组比较，aP<0.05
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表3 4组小鼠全层皮肤缺损创面经GO-GelMA复合水凝胶治疗各时间点创面愈合率比较（%，
组别 样本数 3 d 7 d 14 d 0 μg/mL GO复合水凝胶组 3 22.1±5.5 66.5±8.8 97.1±2.0 0.1 μg/mL GO复合水凝胶组 3 30.8±7.9 66.5±4.9 94.5±1.4 1.0 μg/mL GO复合水凝胶组 3 30.8±9.6 72.4±5.7 97.3±1.0 5.0 μg/mL GO复合水凝胶组 3 33.3±6.4 70.3±4.5 97.8±1.0 F值 1.32 0.89 5.77 P值 0.316 0.434 0.062 注：GO为氧化石墨烯，GelMA为甲基丙烯酸酐化明胶；处理因素主效应，F=2.89，P=0.068；时间因素主效应，F=656.20，P<0.001；两者交互作用，F=1.06，P=0.405；F值、P值为组间各时间点总体比较所得
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表4 4组小鼠全层皮肤缺损创面经GO-GelMA复合水凝胶治疗各时间点MPU比值比较 （
组别 样本数 3 d 7 d 14 d 0 μg/mL GO复合水凝胶组 3 1.77±0.11a 1.29±0.16 1.85±0.22 0.1 μg/mL GO复合水凝胶组 3 2.68±0.16b 2.13±0.24 1.46±0.24d 1.0 μg/mL GO复合水凝胶组 3 1.80±0.08c 1.74±0.17 2.45±0.56 5.0 μg/mL GO复合水凝胶组 3 1.82±0.11a 1.87±0.30 1.75±0.36 F值 30.22 7.83 3.07 P值 0.011 0.093 0.201 注：GO为氧化石墨烯，GelMA为甲基丙烯酸酐化明胶，MPU为平均灌注单位；处理因素主效应，F=2.86，P=0.104；时间因素主效应，F=2.55，P=0.104；两者交互作用，F=8.81，P<0.001；F值、P值为组间各时间点总体比较所得；与0.1 μg/mL GO复合水凝胶组比较，aP<0.01，cP<0.05;与0.1 μg/mL GO复合水凝胶组治疗7 d比较，bP<0.05,dP<0.01
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