Email Alerts
RSS
Volume 38 Issue 7
Jul.  2022
Turn off MathJax
Article Contents
Article Navigation >  CHINESE JOURNAL OF BURNS AND WOUNDS  > 2022  >  38(7): 616-628
Liang LT,Song W,Zhang C,et al.Effects of in situ cross-linked graphene oxide-containing gelatin methacrylate anhydride hydrogel on wound vascularization of full-thickness skin defect in mice[J].Chin J Burns Wounds,2022,38(7):616-628.DOI: 10.3760/cma.j.cn501225-20220314-00063.
Citation: Liang LT,Song W,Zhang C,et al.Effects of in situ cross-linked graphene oxide-containing gelatin methacrylate anhydride hydrogel on wound vascularization of full-thickness skin defect in mice[J].Chin J Burns Wounds,2022,38(7):616-628.DOI: 10.3760/cma.j.cn501225-20220314-00063.
shu

Effects of in situ cross-linked graphene oxide-containing gelatin methacrylate anhydride hydrogel on wound vascularization of full-thickness skin defect in mice

doi: 10.3760/cma.j.cn501225-20220314-00063
  • 1.

    School of Medicine, South China University of Technology, Guangzhou 510006, China

  • 2.

    Research Center for Wound Repair and Tissue Regeneration, Medical Innovation Research Department, the PLA General Hospital, Beijing 100048, China

  • 3.

    Guangdong Cardiovascular Institute, Department of Cardiac Surgery of Guangdong Provincial People's Hospital, Guangdong Academy of Medical Sciences, Guangzhou 510080, China

Funds:

Youth Science Foundation of National Natural Science Foundation of China 32000969, 82002056

Chinese Academy of Medical Sciences Innovation Fund for Medical Sciences 2019-I2M-5-059

Military Medical Innovation Research Project of PLA General Hospital CX19026

Wang Zhengguo Foundation for Traumatic Medicine Growth Factor Rejuvenation Plan SZYZ-TR-03

Key Program of Guangzhou Science Research Plan 201904020047

Special Project of Dengfeng Program of Guangdong Provincial People's Hospital DFJH201812, KJ012019119, KJ012019423

More Information
    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.

     

    • FullText(HTML)
  • loading
    • References(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2019DOI: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.
    • Relative Articles
    • Supplements(0)
    • Cited By
  • 加载中

Catalog

    通讯作者: 陈斌, bchen63@163.com
    • 1. 

      沈阳化工大学材料科学与工程学院 沈阳 110142

    1. 本站搜索
    2. 百度学术搜索
    3. 万方数据库搜索
    4. CNKI搜索

    Figures(10)  / Tables(4)

    Get Citation
    PDF
    XML

    Article Metrics

    Article views (5828) PDF downloads(1355) Cited by()
    Proportional views
    Related

    Copyright © Chinese Journal of Burns京ICP备07035254号-14

    E-mail：shaoshangzazhi@163.com

    Website：https://zhsszz.xml-journal.net/

    Supported by: Beijing Renhe Information Technology Co. Ltd    

    /

    DownLoad:  Full-Size Img  PowerPoint
    Return
    Return