Study On Decellularized Cornea-derived Hydrogels For Promoting Corneal Regeneration And Treating Fungal Infection After Drug Loading

BackgroundThe cornea is the transparent exterior tissue of the eye that covers the pupil,iris and anterior chamber.It protects the eye from the external environment and pathogens,and plays a pivotal role in light transmission and refraction.Many corneal trauma and diseases such as chemical/thermal burns,Stevens-Johnson’s syndrome,ocular cicatricial pemphigoid,corneal ulcers,pseudophakic bullous keratopathy,and Fuch’s endothelial dystrophy can affect corneal transparency,resulting in vision loss,and even blindness.Keratoplasty remains the current gold standard for the treatment of corneal blindness.However,there are more than 1.5 million new cases of corneal blindness are reported every year,of which only less than 5%are treated by corneal transplantations due to donor tissue shortage and the high expense of transplantation surgery.Therefore,the development of a suitable alternative graft material to solve the shortage of donor cornea for transplantation has become the focus of increased attention.The successful development of decellularized porcine cornea(DPC)tissue engineering biomaterials has alleviated the shortage of corneal donors to some extent.However,in some corneal dysfunctions,the infections or injury occurs within a well-defined focal area,leaving the surrounding corneal tissue healthy and intact.In these cases,replacing only the damaged tissue would be attractive as the use of DPC grafts requires extensive and multiple invasive surgeries.Tissue engineering provides a new promising approach by which a scaffold can be designed for minimally invasive aesthetic and functional reconstruction of damaged tissues.Hydrogels,which comprise three-dimensional networks of polymer,allowing for the diffusion of oxygen,water and glucose through their networks in hydrated state,and their physicochemical characteristics can simulate the cell’s natural microenvironment to promote wound healing.In addition,the hydrogel has the following advantages:capacity to be injected with minimally invasive surgery to form a hydrogel in situ;ability to fill in tissue defects of different sizes and geometries;regulable mechanical properties that can be controlled by changing the concentration or strengthened by cross-linking;easily modification that can be augmented with cells,growth factors and drugs;good processibility that allow fabrication of customized structures through three-dimensional(3D)printing.Recently,decellularized porcine and bovine cornea-derived hydrogels have been prepared for corneal use,and they have been demonstrated to be biocompatible in vitro and in vivo.However,the reported decellularized corneal hydrogel can only gel at 37℃,and the prepared hydrogel is opaque.More importantly,the ability of these hydrogels to repair damaged cornea has not been deeply studied.Fungal keratitis is an intractable ophthalmic disease.The therapies against fungal keratitis are through topical or systemic medical therapy alone or in combination with surgical treatment.Antifungal eye drops is a non-invasive,economical and convenient drug delivery system,which has always been considered as the most commonly used treatment method.However,quite often,this method is often low in bioavailability due to short contact time on the ocular surface(within 1-2 min).In fact,only 1-7%of the eye drops can reach the focus area,while the rest is cleaned up by eye’s movement and the scouring nasolacrimal system.Surgical interventions are required for uncontrollable or further aggravated keratitis infection,such as corneal debridement,conjunctival flaps,lamellar keratoplasty(LK)and penetrating keratoplasty(PK).However,for the above mentioned fungal infections that occur in specific areas of the cornea and involve the stroma,corneal debridement will leave scars,conjunctival flap covering will affect the aesthetics and vision,and corneal transplantation is costly and invasive.Therefore,it is urgent to develop a new drug delivery system that can control the release of antifungal drugs and promote corneal defects regeneration.Herein,in this study,we prepared a transparent DPC-derived hydrogel,which can gel at room temperature for promoting the repair of focal corneal epithelial and stromal defects,and for the treatment of corneal fungal infection after drug delivery.Our study was divided into the following three parts:(1)Preparation and characteristic analysis of DPC-derived hydrogel;(2)Study on the DPC-derived hydrogel for the regeneration of epithelium and stroma in focal corneal defects;(3)Study on the DPC-gelatin hydrogels hybrid voriconazole loaded microspheres for ocular antifungal treatment.Part Ⅰ Preparation and characteristic analysis of DPC-derived hydrogelObjectiveThe DPC-derived hydrogel has many excellent biological properties.The purpose of this section is to study the preparation and characterization of DPC-derived hydrogel.Methods1.Preparation of DPC.Fresh pig eyes were thoroughly rinsed with autoclaved phosphate buffer saline(PBS)and the epithelium was scraped off by a corneal epithelial scraper.The central cornea was drilled through a 10 mm ring drill,and the endothelium was scraped off.The porcine cornea was then treated with decellularized fluid containing 0.5%sodium lauroylglutamate and 500 U/mL of recombinant endonuclease.2.Preparation of DPC-derived hydrogel.The lyophilized DPCs were milled into a fine powder,fully digested with 0.1N HCl solution containing pepsin.Added with 1 N NaOH to adjust the pH value to 7.4 after complete digestion,then added with a crosslinking agent N-cyclohexyl-N’-(2-morpholinoethyl)carbodiimide metho-p-toluenesulfonate(CMC)and N-hydroxysuccinimide(NHS)for crosslinking,and placed at room temperature for 15-20 min to from the hydrogel.3.Biological characterization of DPC-derived hydrogel.Including gel time,transparency,in vitro light transmittance,ultrastructure,DNA residue,a-gal content,glycosaminoglycan(GAG)content,collagen content and rheological properties.Live/dead staining,cell counting Kit-8(CCK-8)assay,F-actin staining and scanning electron microscopy(SEM)observation were used to detect the effect of hydrogels on the proliferation activity of human corneal fibroblasts(HCFs)and human corneal epithelial cells(HCECs)to evaluate their in vitro biocompatibility.ResultsThe DPC-derived hydrogel can be glue for 15-20 min at room temperature,and has excellent transparency and light transmittance.SEM showed that it had a porous surface structure.No significant antigenic component DNA and α-gal residues were observed,while the bioactive components GAG and collagen were effectively retained.The hydrogel exhibited distinct mechanical properties,possessed good biocompatibility,and has no obvious effect on the proliferation activity of HCFs and HCECs.Conclusion1.The DPC-derived hydrogel has the advantages of injectability,rapid gelation,good transparency and biocompatibility.2.The DPC-derived hydrogel may be an ideal biomaterial to promote the repair of corneal epithelial and stromal defects.Part Ⅱ Study on the DPC-derived hydrogel for the regeneration of epithelium and stroma in focal corneal defectsObjectiveThe aim of this study was to test the in vitro functional of DPC-derived hydrogel,and in vivo biocompatibility and functionality was further verified to evaluate its feasibility in repairing corneal epithelium and stroma defects.Methods1.Evaluating the in vitro functional of DPC-derived hydrogel.The HCFs in the experimental group were inoculated on the well plate coated with the DPC-derived hydrogel,while the HCFs in the control group were inoculated directly on the blank well plates.Sirius red assay,immunofluorescence staining,qRT-PCR and Western blotting analyses were performed to assess the difference in the expression of the relative protein and gene of HCFs on hydrogel and blank well plates,including keratocan,lumican,decorin,collagen and α-SMA.2.In vivo application of DPC-derived hydrogel.The focal epithelial and stromal defects of rabbit cornea with 1/3 full thickness were prepared by a 3.5 mm trephine.The rabbit cornea in the experimental group was applied with the DPC-derived hydrogel,while the control group cornea was not treated with hydrogel,and the other operations were the same as those in the experimental group.At 1 week after surgery,slit-lamp microscopy and fluorescence staining were performed to evaluate the defect area of the corneal epithelium in two groups.Slit-lamp microscopy,anterior segment-optical coherence tomography(AS-OCT)and confocal microscope were conducted at 4 and 12 weeks after surgery to observe the corneal transparency,corneal epithelium and stromal thickness,and stromal turbidity in the two groups.3.Histological analysis.After 4 and 12 weeks of surgery,rabbits’ eyeballs in the two groups were removed respectively,and changes of corneal epithelium shape,thickness,stromal scar and thickness were observed by hematoxylin and eosin(H&E)staining.Immunohistochemical(IHC)staining was used to detect the expression of scar formation marker α-SMA.Results1.The in vitro functional of DPC-derived hydrogel.Sirius red assay,immunofluorescence staining,qRT-PCR and Western blotting analysis results revealed high expression of the keratocyte marker keratocan and the corneal proteoglycans lumican and decorin,but low expression of the myofibroblast-associated markers collagen I and α-SMA in HCFs on hydrogels compared with that observed in cells on blank well plates.2.In vivo biocompatibility and biointegration of DPC-derived hydrogel.Fluorescein staining images were obtained using a cobalt blue slit-lamp.The results revealed that the defect area of the corneal epithelium gradually decreased over time in the hydrogel-treated group(less than 3 days to re-epithelialize),while re-epithelialization spanned 5-7 days in the control group.The in vivo slit-lamp,AS-OCT and confocal microscopy imaging results demonstrated that the levels of corneal stromal haze and scarring were greater 4 weeks after surgery in the control group than those observed in the hydrogel-treated group.The corneas were almost completely transparent 12 weeks after application of the hydrogels.The confocal microscopy imaging and H&E staining results further showed that the morphology and arrangement of the regenerated corneal epithelium in the hydrogel-treated group were not significantly abnormal,the thickness of the epithelium was not significantly different from that of the normal corneal epithelium,and the stroma thickness was within the range of the normal corneal matrix thickness without obvious scar.However,the corneal epithelial hyperplasia,stroma opacity,scar hyperplasia and positive expression of α-SMA were observed in the control group 4 weeks after surgery.Conclusion1.The DPC hydrogel may provide a microenvironment similar to that of the normal cornea to support cell growth and promote extracellular matrix(ECM)synthesis.2.The presence of the hydrogel may inhibit fibrosis reactions early on and reduce the effect of scar repair.3.In terms of the in-situ gelling and injectable characteristics of the DPC hydrogel,it is expected to realize the treatment of focal corneal defect without lamellar keratoplasty,and reduce rejection and treatment cost.Part Ⅲ Study on the DPC-gelatin hydrogels hybrid voriconazole loaded microspheres for ocular antifungal treatmentObjectiveIn this study,we focused on the preparation of the Vor-loaded MCs followed by a detailed characterization.The DPC-gelatin hydrogel was used as a sustained drug delivery system to evaluate the feasibility of the repair of focal corneal defect while treating fungal infection.Methods1.Synthesis of Vor-loaded MCs.Vor-loaded MCs was prepared by emulsion solvent evaporation.A certain amount of poly-lactic-co-glycolic acid(PLGA)and Vor are dissolved in different proportions of dichloromethane and acetone(1:1,1:4,4:1)(oil phase).Add water phase containing emulsifier polyvinyl alcohol(PVA)drop wise into the oil phase,and ultrasonically emulsifying with an ultrasonic pulverizer to obtain emulsion.The emulsion was added to the external water phase,and stirring with a magnetic stirrer to remove the organic solvents completely.Then,centrifugated by a high-speed cooling centrifuge,and freeze-dried to obtain the Vor-loaded MCs.2.Characterization of Vor-loaded MCs.The particles size,polydispersity index(PDI),zeta-potential,drug loading,encapsulation efficiency,morphology,fourier transform infrared(FTIR)spectrum and differential scanning calorimetry(DSC)of the resulting MCs were assessed.3.Preparation of MCs-loaded DPC-gelatin hydrogels.The lyophilized DPCs were milled into a fine powder,fully digested by a mixed liquid containing 0.1 N HCl and pepsin.Subsequently,the pH was adjusted to 7.4 with 0.1 N NaOH,and finally lyophilized to obtain dry prepolymer DPC hydrogel powders.Then,a certain amount of the lyophilized prepolymer DPC hydrogel powders,gelatin and MCs were dissolved in Milli-Q water,and cross-linked to synthesize MCs-loaded DPC-gelatin hydrogels.4.Biological characterization of MCs-loaded DPC-gelatin hydrogels.Including surface morphology,rheological characteristics,in vitro biodegradation,in vitro drug release behavior.Live/dead staining and CCK-8 assay were used to detect the effect of MCs-loaded DPC-gelatin hydrogels on the proliferation activity of HCFs and HCECs to evaluate their in vitro biocompatibility.5.In vitro antifungal activity studies on MCs-loaded DPC-gelatin hydrogels.(1)The zone of inhibition studies:The DPC-gelatin hydrogels,Vor,MCs,and the MCs-loaded DPC-gelatin hydrogels were aseptically placed at the center of the Petri dish coated with F.solani and A.fumigatus.After 24 h of incubation,the diameter of inhibition zone was measured with a Vernier caliper.(2)Live/dead assay:The DPC-gelatin hydrogels and the MCs-loaded DPC-gelatin hydrogels were put into EP tubes containing F.solani and A.fumigatus,respectively.A live and dead cell double staining kit was used to determine the fungal viability after 24 h of incubation.(3)Ex vivo culture of rabbit eyeball infected by fungi:A 5.0 mm corneal trephine was applied on the central cornea of fresh rabbit eyeballs to remove certain part of corneal epithelium-stroma(approximately 1/2 corneal thickness).Subsequently,a certain concentration of F.solani suspension was inoculated directly on the corneal defect.After 24 h of fungal incubation,infected ex vivo corneas were randomly divided into three groups,which were the MCs-loaded DPC-gelatin hydrogels treatment group,the DPC-gelatin hydrogels treatment group,and blank control group.Normal corneas without any treatment were negative control group.Central corneas were harvested using an 8.0 mm corneal trephine after hydrogel application for 24 h,and colony counting,calcofluor white fluorescence and H&E staining were performed to assessment antifungal efficiency of the MCs-loaded DPC-gelatin hydrogels.Results1.Characterization of Vor-loaded MCs.The MCs fabricated with DCM:acetone ratio(4:1)demonstrated the smallest particle size(260.13 ± 61.76 nm),the highest drug loading percentage and entrapment efficiency,which are 3.66 ± 0.44%and 40.67 ±4.83%,respectively.SEM images revealed that the MCs(4:1)are morphology and have a smooth surface.The FTTR absorption spectra and DSC thermograms indicated the complete encapsulation of the Vor.2.Biological characterization of MCs-loaded DPC-gelatin hydrogels.SEM imaging,in vitro biodegradation and the rheological behavior analysis showed that the addition of Vor-loaded MCs did not affect the porous structure,rheological properties and in vitro biodegradation profile of the DPC-gelatin hydrogels.In vitro drug release studies showed that there was a sustained release of Vor from MCs for 7 days,with almost～50%was released within 2 days and～80%within 3 days.The Vor release profiles from MCs incorporated in DPC-gelatin hydrogel were similar to the Vor release profiles from MCs.The results of Live/dead staining and CCK-8 assay showed that the MCs-loaded DPC-gelatin hydrogels had no obvious effect on the proliferation activity of HCFs and HCECs,and had good biocompatibility.3.In vitro antifungal activity of MCs-loaded DPC-gelatin hydrogels.The results of zone of inhibition studies indicated that the MCs and MCs-loaded DPC-gelatin hydrogels could also hinder the proliferation of F.solani and A.fumigatus in comparison with the formulations comprising just the free drug.Live/dead staining and ex vivo culture of rabbit eyeball infected by fungi datas demonstrated that MCs-loaded DPC-gelatin hydrogels could effectively inhibit the proliferation of fungi,while the DPC-gelatin hydrogels alone has no antifungal effect.Conclusion1.The new MCs-loaded DPC-gelatin hydrogels drug delivery system successfully overcomes the disadvantages of traditional treatment methods such as low bioavailability of eye drops,scar left by surgery,or aesthetic impact,or high cost and invasive of surgery.2.The new MCs-loaded DPC-gelatin hydrogels drug delivery system has excellent antifungal activity and could promote the repair of focal corneal defect,which is expected to be used in the treatment of fungal keratitis with focal corneal defect.