Construction And Application Of Graphene Oxide And Niobium Carbide MXene Nanocomposite For Artificial Cornea Scaffold

Backround:Corneal disease is one of the most common blindness-related eye diseases approximately 25%of blind worldwide.Approximately 4,000,000 people with corneal blindness in either one or both eyes live in China with 200,000 new blind patients added each year,whereas corneal transplants is much lower than 1/400 the total number of corneal blindness.The lack of donor corneal material remains the largest limiting factor in current corneal transplantation surgery.The graft failure rate after penetrating keratoplasty was 20%for the first surgery,multiple surgeries lead to a higher graft failure rate,and even no chance to surgery.It is previously known that corneal transplant surgery was not amenable to be successful in patients of end-stage corneal diseases.The advent of artificial cornea offers the only hope to them.Moreover,there is a variety of artificial cornea models for corneal applications,but it is generally known that complications after implantation,thus contributing to undesired effects and has not been in wide-spread use.Low biocompatibility,optical zone and artificial cornea scaffold were not connected tightly,scaffold dissolved or absorbed and surgical procedure was complex lead to the occurrence of complications,such as low tissue integration,aqueous humor leakage,scaffold dislodgement,endophthalmitis,glaucoma and retinal complication.The 2D niobium carbide（Nb₂C）is a novel kind of MXene material with outstanding mechanical properties,excellent stability against high temperature and resistance to chemical resistance,good fastness to rubbing,and good mechanical stability,which can be used to make an ideal corneal scaffold.Meanwhile,Graphene oxide（GO）is a new kind of two-dimensional carbon nanomaterial,which is gaining substantial attention due to the unique two-dimensional honeycomb structure and excellent properties,and promote cell proliferation and differentiation.Studies utilizing novel biomaterials and technology to make an ideal artificial corneal with biological activity have not yet been reported.In order for the scaffold to robust integration with the host tissue,bioactive molecules acting synergically improve the biomechanical properties of the scaffold and generate a synergistic effect.Therefore,based on the requirements of ideal artificial cornea scaffold,this project aims to take advantage of unique biological activity and good mechanical features in synthesis and applications of GO and Nb₂C nanocomposite for making an artificial cornea scaffold with biological activity,safety and efficacy to strengthen and explore the applications in artificial cornea.Method:1.The Nb₂C was synthesized via HF etching and TPAOH intercalating.Then,Nb₂C nanofibrous scaffold with a relatively smooth surface was prepared by calendering treatment and scraping-coating methods.The elemental composition of Nb₂C nanofibrous scaffold,morphology and structure were characterized by various techniques.The biocompatibility of Nb₂C nanofibrous scaffolds was investigated by acute systemic toxicity test,haemolytic test and cytotoxicity test,etc.2.Graphene oxide（GO）and Nb₂C were purposively selected for this section.The different mass ratio of GO/Nb₂C nanocomposite scaffolds were prepared by calendering treatment and scraping-coating methods,meanwhile,they were characterized via SEM,XRD and XPS.Through characterization of the morphology,structure,surface wettability and mechanical properties,the performance differences of GO/Nb₂C nanocomposite scaffolds with different weight ratios were systematically compared.Meanwhile,taking corneal fibroblasts as a target cell,the effect of nanocomposite scaffolds on the in vitro biocompatibility and cell behaviour was evaluated via Calcein-AM assay,cell apoptosis analysis,cell proliferation test,and cell electron microscopy.3.The different mass ratio of GO/Nb₂C nanocomposite scaffolds with biological activity were implanted into cornea of New Zealand white rabbits to evaluate the effects of the scaffolds in rabbit corneas.Anterior segment of picture,anterior segment of picture OCT（AS-OCT）,in vivo confocal microscopy,HE staining and Masson staining were used to observe the cornea.Fibrosis indexes heat shock protein 47（Hsp47）,Fibronectin（FN）、a-smooth muscle actin（a-SMA）were examined by Immunofluorescence（IF）and immunohistochemistry（ICH）.Inflammatory indexes CD11b,interleukin 2（IL-2）and interleukin 6（IL-6）were examined by IF and Enzyme-linked immunosorbent assay（ELISA）.Oxidative stress indexs malondialdehyde（MDA）,superoxide dismutase（SOD）and glutathione peroxidase（GSH-Px）were examined.Investigate the effect of GO/Nb₂C nanocomposite scaffolds on the rabbit cornea and determine the optimized mass ratio of GO/Nb₂C.4.One-piece model of artificial cornea was designed.The 0.5GO/Nb₂C nanocomposite scaffold and PMMA were chosen as the artificial cornea scaffold and optical cylinder,respectively.Meanwhile,the rabbit corneal alkali burn model was established in vivo.The one-piece model of artificial cornea was implanted in vivo alkali burn-injured cornea model and the effects were observed by anterior segment of picture,HE staining and Masson staining,IF and ICH.Resultes:1.The Nb₂C and Nb₂C nanofibrous scaffold with a relatively smooth surface were successfully synthesized via HF etching,TPAOH intercalating,and calendering treatment and scraping-coating methods.Acute toxicity test showed that unusual presentation such as manic episodes and sleep disruption were not observed in either the experimental or control groups.No significant differences in weight were noted between the two groups after injection 4 h、24 h、48 h and 72 h（P>0.05）.Haemolysis was 0.49%,which showed low haemolysis less than5%.HK have around 98%survival rates with excellent growth in either the experimental or control groups and no significant differences were observed（P>0.05）.2.XRD and other basic physicochemical properties showed that GO/Nb₂C nanocomposite scaffolds were successfully prepared.After the incorporation of GO,the contact angle and modulus of elasticity of nanocomposite scaffolds were reduced,hydrophilicity,porosity,and modulus of elasticity were increased.Corrosion behaviour test in artificial tear fluid showed excellent corrosion.The HK cell activity rate was more than 98%in all GO/Nb₂C nanocomposite scaffolds,and there was no difference compared with the control group（P>0.05）.Cells proliferated equally well on the material with 72%～83%cell proliferation index.Compared with the control,GO/Nb₂C nanocomposite scaffolds could promote the proliferation to a certain extent.The apoptosis rates of GO/Nb₂C nanocomposite scaffolds were lower than 5%in all groups and there were no significant differences among the groups（P>0.05）.The electron microscopy images obtained revealed that cells adhered and spread over the material,nanocomposite scaffolds provide more benefits for cell adhesion and growth compared with pure Nb₂C.The preliminary optimized 0.5GO/Nb₂C was determined based on the better mechanical properties and in vitro biocompatibility.3.The results presented in vivo were shown as follows:the cornea tissue appeared to be morphologically unaffected via HE and Masson staining,anterior segment OCT（AS-OCT）and corneal confocal microscopy,without obvious fibrous hyperplasia and inflammatory cell infiltration.Fibrosis index Hsp47,FN,a-SMA are negative in rabbit cornea.Inflammation index CD11b is negative,IL-2 and IL-6 is downregulated,meanwhile.Oxidative stress indexs are downregulated.The GO/Nb₂C nanocomposite scaffolds showed effect of antioxidative stress and anti-inflammatory property.The above results demonstrated that GO/Nb₂C nanocomposite scaffold had stable nature,good biocompatibility,and excellent mechanical properties,which could be a promising candidate for artificial cornea and provide a favorable microenvironment for cornea.The0.5GO/Nb₂C group showed that the anti-corrosion ability,hydrophilicity,and mechanical properties is slightly well than the other group,which was determined as the optimized ratio in vivo.4.The artificial cornea was not removed and it remained in place,and no keratolysis,endophthalmitis,and glaucoma were observed two months after the procedure stage I.One of the cases developed corneal thinning one month after the procedure stage II.No precorneal proliferative membranes was observed two months after the procedure stage II.Conclusions:1.GO and Nb₂C nanocomposite for artificial cornea scaffold was prepared,thus achieving good mechanical and biocompatibility.The in vivo biocompatibility and bioactivity of new MXene materials GO/Nb₂C nanocomposite scaffold was firstly examined,which broadens the scope of Nb₂C applications in the biomedical field.2.The nanocomposite scaffold is low cytotoxicity,a trend of enhances the cell proliferation,and good cell integration in vitro,good tissue integration,no corneal fibrosis,antioxidative stress and anti-inflammatory property in vivo as well.3.The moderate or severe burns rabbit corneal alkali burn model was successfully established in vivo with sodium hydroxide（1mol/L）in circular filter paper（?=10mm）for 40s.One-piece model of artificial cornea can be used to treat corneal alkali burn with the potential recovery of visual function.4.GO/Nb₂C nanocomposite has extremely high biocompatibility with biological activity,safety and efficacy and is a promising bioactivity corneal scaffold for an artificial cornea providing new methods and ideas for the construct functional artificial cornea scaffold and one-piece model of artificial cornea future development.In summary,based on the current hot spots,GO/Nb₂C nanocomposite scaffold and one-piece model of artificial cornea were designed by combining the unique physicochemical properties of graphene,respectively.The potential of scaffolds and one-piece model of artificial cornea in cornea were investigated in vitro and in vivo.Our research provides new directions and ideas for the future development of artificial cornea.