Artificial cornea preparation using collagen/chondroitin sulfate/fibroblast growth factor composite film

doi:10.3969/j.issn.2095-4344.0767

Abstract

Abstract:

BACKGROUND: The traditional corneal scaffolds exhibit poor strength and biological compatibility. Little is reported on the artificial cornea prepared by collagen and chondroitin sulfate (CS), which consist of the natural corneal tissue.

OBJECTIVE: To prepare the collagen/CS/fibroblast growth factor (FGF) composite artificial cornea with slow-release growth factor, high strength and light transmittance, as well as good biocompatibility.

METHODS: Regenerated collagen films were prepared by 1%, 5%, 10% collagen solutions using flow casting method, and the regenerated collagen film with the best bioactivity that was prepared by 5% collagen solution was screened through a biomechanical test. Then, the CS/collagen composite film was achieved by cross-linking the CS (2, 20, 80 g/L) with collagen by using N-(3-Dimethylaminopropyl)- N’S-ethylcarbodiimide hydrochloride-N-Hydroxysuccinimide. The composite film made of 20 g/L CS was confirmed to have the best transparency, which was used to be mixed with 5, 25, 50 mg/L FGF in PBS for 24 hours to prepare the collagen/CS/FGF composite films. ELISA method was used to detect the FGF level in the supernatant. Afterwards, corneal epithelial cells were co-cultured with regenerated collagen film, collagen/CS composite film and collagen/CS/FGF composite film, respectively. After 48 hours of co-culture, cell proliferation was detected by MTT method, based on which we could screen the optimal collagen/CS/FGF composite film. After co-culture with the collagen/CS/FGF composite film for 48 and 72 hours, cell morphology was observed by confocal microscope and scanning electron microscope, respectively.

RESULTS AND CONCLUSION: The release amount of FGF from the composite films was dependent on the initial loading amount of FGF. Meanwhile, FGF released slowly from the three kinds of composite films, and the release amount was 11%, 23%, 30% at 72 hours after culture, in accordance with the pharmacokinetic process. MTT findings indicated that the optimal loading concentration of FGF was 25 mg/L. Under the microscope, the collagen/CS/FGF composite film promoted the adhesion, growth and proliferation of corneal epithelial cells. To conclude, the collagen/CS/FGF composite film is expected to be an ideal scaffold material for artificial cornea preparation.

中国组织工程研究杂志出版内容重点：生物材料；骨生物材料; 口腔生物材料; 纳米材料; 缓释材料; 材料相容性；组织工程

Key words: Collagen, Cornea, Dermatan Sulfate, Tissue Engineering

CLC Number:

R318

Mao Bao-liang, Hu Bin, Jia Lei, Shan Heng-yue, Li Xiang, Wang Ying, Yuan Wan-juan, Zhang Feng-chao, Chen Jing-hua . Artificial cornea preparation using collagen/chondroitin sulfate/fibroblast growth factor composite film[J]. Chinese Journal of Tissue Engineering Research, 2018, 22(14): 2203-2208.

Figures/Tables 7

References

[1] Mirabelli P,Mukwaya A,Lennikov A,et al.Genome-wide expression differences in anti-Vegf and dexamethasone treatment of inflammatory angiogenesis in the rat cornea.Sci Rep.2017;7(1):7616.

[2] Meek KM,Knupp C.Corneal structure and transparency.Prog Retin Eye Res.2015;49(2):1-16.

[3] Stafiej P,Kung F,Thieme D,et al.Adhesion and metabolic activity of human corneal cells on PCL based nanofiber matrices. Mater Sci Eng C Mater Biol Appl.2017;71:764-777.

[4] Riau AK,Mondal D,Yam GHF,et al.Surface modification of PMMA to improve adhesion to corneal substitutes in a synthetic core-skirt keratoprosthesis.ACS Appl MaterInterfaces.2015;7(39): 21690-21702.

[5] Hemalatha T,Yadav S,Krithiga G,et al.Chitosan as a matrix for grafting methyl methacrylate: synthesis, characterization and evaluation of grafts for biomedical applications.Polym Bull(Berl). 2016;73(11): 3105-3117.

[6] Yawon H,Gonhyung K.Evaluation of stability and biocompatibility of PHEMA-PMMA keratoprosthesis by penetrating keratoplasty in rabbits. Lab Anim Res.2016;32(4):181-186.

[7] 李明.多孔n-HA/PVA/CF(PEEK)复合水凝胶的制备与性能研究[D].深圳:深圳大学化学与化工学院,2015:1-86.

[8] Kharaghani D,Meskinfam M,Rezaeikanavi M,et al.Synthesis and characterization of hybrid nanocomposite via biomimetic method as an artificial cornea.Invest Ophth Vis Sci.2015;56(7):5024.

[9] Chaiyasan W,Praputbut S,Kompella UB,et al.Penetration of mucoadhesive chitosan-dextran sulfate nanoparticles into the porcine cornea.Colloids Surf B Biointerfaces.2017;149:288-296.

[10] Huang X,Wang R,Lu T,et al.Heparin-like chitosan hydrogels with tunable welling behavior, prolonged clotting times, and prevented contact activation and complement activation.Biomacromolecules. 2016;17(12):4011-4020.

[11] Jamard M,Hoare T,Sheardown H.Nanogels of methylcellulose hydrophobized with N-tert-butylacrylamide for ocular drug delivery. Drug Deliv Transl Res.2016;6(6):648-659.

[12] Tummala GK,Joffre T,Lopes VR,et al.Hyperelastic nanocellulose- reinforced hydrogel of high water content for ophthalmic applications. ACS Biomater Sci Eng.2016;2(11):2072-2079.

[13] Sepulveda RV,Valente FL,Reis ECC,et al.Bacterial cellulose and bacterial cellulose/polycaprolactone composite as tissue substitutes in rabbits' cornea.Pesquisa Vet Brasil.2016;36:986-992.

[14] Patchan MW,Chae JJ,Lee JD,et al.Evaluation of the biocompatibility of regenerated cellulose hydrogels with high strength and transparency for ocular applications.J Biomater Appl. 2015;30(7):1049-1059.

[15] Rudisill SG,DiVito MD,Hubel A,et al.In vitro collagen fibril alignment via incorporation of nanocrystalline cellulose.Acta Biomaterialia.2015; 12(1):122-128.

[16] Zhao X,Song W,Liu S,et al.Corneal regeneration by utilizing collagen based materials.Sci China-Chem.2016;59(12):1548-1553.

[17] Mohamed-Noriega K,Butron-Valdez K,Vazquez-Galvan J,et al.Corneal melting after collagen cross-linking for keratoconus in a thin cornea of a diabetic patient treated with topical nepafenac: a case report with a literature review.Case Rep Ophthalmol.2017;7(1):119-124.

[18] Zhao X,Long K,Liu Y,et al.To prepare the collagen-based artificial cornea with improved mechanical and biological property by ultraviolet-A/riboflavin crosslinking.J Appl Polym Sci.2017;134(38): 45226.

[19] Medeiros CS,Giacomin NT,Bueno RL,et al.Accelerated corneal collagen crosslinking: Technique, efficacy, safety, and applications.J Cataract Refract Surg.2016;42(12):1826-1835.

[20] Hu C,Yu L,Wei M.Biomimetic intrafibrillar silicification of collagen fibrils through a one-step collagen self-assembly/ silicification approach.RSC Adv.2017;7(55):34624-34632.

[21] 刘洁,赵世玉,徐洲,等.胶原在两种离子液体[BMIM]Cl 和[BMIM]Ac 中的溶解及再生结构比较[J].化工学报,2015,66(6):2196-2204.

[22] Li C,Liao H,Zhang X,et al.Preparation of cationic modified collagen extracted from leather wastes and their application in dye flocculation.J Appl Polymr Sci.2017;134:45363.

[23] Younesi M,Donmez BO,Islam A,et al.Heparinized collagen sutures for sustained delivery of PDGF-BB: delivery profile and effects on tendon-derived cells in-vitro.Acta Biomaterialia.2016;41:100-109.

[24] Hariya T,Tanaka Y,Yokokura S,et al.Transparent, resilient human amniotic membrane laminates for corneal transplantation.Biomaterials. 2016;101:76-85.

[25] Chae JJ,Choi JS,Lee JD,et al.Physical and biological characterization of the gamma-irradiated human cornea.Cornea. 2015;34(10):1287-1294.

[26] Pospichal R,Nesmerak K,Rychlovsky P,et al.Determination of chondroitin sulfate by thiazine dyes using flow injection analysis with spectrophotometric detection.Anal Lett.2007;40(6):1167-1175.

[27] Ribeiro AM,Figueiras A,Veiga F.Improvements in topical ocular drug delivery systems: hydrogels and contact lenses.J Pharm Pharm Sci. 2015;18(5):683-695.

[28] Cai J,Dou G,Zheng L,et al.Pharmacokinetics of topically applied recombinant human keratinocyte growth factor-2 in alkali-burned and intact rabbit eye.Exp Eye Res.2015;136:93-99.