Bio-modulation of scaring Glaucoma Filtration Surgery using a novel application of coated magnesium

Xingji Li,Rihrd Filek,Xiomin Zhu,Hunhun Go,Liying Qio,Hong Liu,Lin Xie,?,Yong Wng,?,Fusheng Pn,Cindy ML Hutnik,?

a Department of Ophthalmology,Institute of Surgery Research,Daping Hospital,Army Medical University,Chongqing 400042,China

b Department of Ophthalmology,Western University,Ivey Eye Institute,St.Joseph’s Health Care London,London,ON,Canada

c Department of Ophthalmology,The Third Affiliated Hospital,Chongqing Medical University,Chongqing 400016,China

d Collage of Material Science & Engineering,Chongqing University,Chongqing 400045,China

eNational Engineering Research Center for Magnesium Alloys,Chongqing University,Chongqing 400045,China

Received 3 December 2019;received in revised form 15 February 2020;accepted 23 February 2020

Available online 3 October 2020

Abstract

Keywords:Glaucoma;Glaucoma drainage device;Fibrosis;Tenon’s capsule fibroblasts;Magnesium;Coating.

1.Introduction

The World Health Organization ranks glaucoma as the second most common cause of blindness,and the leading cause of irreversible blindness[1,2].Since 2010 there are about 60.5 million patients worldwide who suffer from primary open angle glaucoma(POAG)and primary angle-closure glaucoma(PACG),and the amount is estimated to increase to 79.6 million by 2020[3].Filtration surgery still plays a mainstream role of treatment for glaucoma.In order to avoid formatted bleb and obstruction of the fistula,the inhibition of cell proliferation and inflammation is often necessary and desirable after filtering surgery.In filtering surgery,the most frequently used substances are mitomycin C(MMC),and 5-fluorouracil(5-FU).These antimetabolites improve the success rate for long-term intraocular pressure(IOP)control and reduce the risk of bleb failure[4,5],but their use is associated with an increased complications including thin-walled blebs,hypotony,wound leakage and infection[6–9].Until now,no satisfactory methods have been developed to resolve this issue.

In recent years,filtration surgery has undergone various modifications with trabeculectomy no longer being the only choice.There is an increasing number of glaucoma patients being now treated by glaucoma drainage devices and implantations.Lowering IOP can be achieved by increasing aqueous humour drainage through this artificial route.Currently,permanent and inert metals like titanium alloy are used in the main types of glaucoma implants,such as Ex-press,iStent,and the CyPass Micro-Stent implant.However,this material is associated with long-term complications because the permanent foreign body induces inflammation under the tenon’s capsule.The initial process of inflammation is characterized by increased numbers of monocytes and lymphocytes within the early proliferation of connective tenon’s capsule tissue[10].The main risk factor includes fibroblast proliferation[11–13],development of filtering bleb and fistula scarring[14,15].

An inflammatory response against a foreign body is a natural defense process,which limits the development of permanent metal implants[16,17].The foreign body may associate with subconjunctival inflammation due to toxicity and an allergic response.The inflammatory phase is characterized by the activation of the innate immune system and the release of inflammatory cytokines.Chronic inflammation is an established risk factor for fibrosis due to the release of mediators such as interleukin-1(IL-1)and tumor necrosis factor-alpha(TNF-α)which lead to increased tissue levels of transforming growth factor-beta(TGF-β).Chronic inflammatory reactions associated with a foreign body can accelerate wound scaring,resulting in glaucoma surgery failure.The success rate would be higher if proliferation was prevented by an inhibitory substance that remained in contact with the tissue.In addition,the implemented material should be biodegradable to circumvent the inflammatory stimulation.

The use of magnesium is advantageous due to its biodegradability,adjustable mechanical strength,light weight,and high biological compatibility[18].Coated magnesium has been considered a suitable biomaterial for glaucoma valve implant surgery.Various studies have found that magnesium is biologically safe and have elucidated positive biological reactions in ocular tissue[19].Magnesium is an essential element for the human metabolism,being the second most common intracellular cation.Magnesium also plays a critically role for maintaining the functional and structural integrity of several vital ocular tissues such as the cornea,lens and retina[20].Magnesium is a calcium blocker[21]and increases blood flow to tissues through endothelin-1 and endothelial nitric oxide pathways[22].In addition,magnesium has been shown to play a neuroprotective role in glaucoma patients[23,24]by preventing oxidative stress and apoptosis[25].However,magnesium has poor corrosion resistance,which becomes a major obstacle to their applications as an implant.Having a coating on the magnesium is an effective way to improve the corrosion resistance.

Preliminary data indicates that coated magnesium may potentially inhibit human tenon’s capsule fibroblasts(HTCFs)growth and proliferation.The purpose of this study is to evaluate the in-vitro biocompatibility and anti-proliferative properties of differently coated magnesium in a primary culture of human tenon’s capsule fibroblasts(HTCFs)and in rabbit Glaucoma Filtration Surgery.

2.Methods

2.1.Fabrication of differently coated magnesium

Magnesium with a purity of 99.99%was obtained from the National Engineering Research centre for Magnesium Alloys,Chongqing University,China.The as-cast pure magnesium was extruded to 14.5mm in diameter at 200°C with an extrusion ratio of 25,and then cut into 1mm thickness disks.The surface of the disks was polished using 320–800#abrasive paper.All of the disks were ultrasonically cleaned in acetone,air dried and weighed.The magnesium disks were coated with either hydroxyapatite(HA,2μm in thickness),dicalcium phosphate dihydrate(DCPD,3μm in thickness),or DCPD+stearic acid(DCPD+SA,3μm in thickness).

All the coatings were fabricated by solution deposition processes.The HA coating was deposited in the aqueous solution containing 0.25mol/L ethylenediaminetetracetic acid calcium disodium salt hydrate(EDTA-Ca)and 0.25mol/L potassium dihydrogenphosphate(KH2PO4),and the DCPD coating in 3.1% sodium hydrogen phosphate(Na2HPO4)and 5.3%calcium nitrate(Ca(NO3)2).The HA and DCPD coatings were formed with a chemical bond of calcium phosphate and magnesium matrix.The DCPD+SA coating was obtained by immersing the DCPD-coated magnesium disks in ethanol solution of 0.5mol/L stearic acid.The combination of SA depends on hydrogen bond and physisorption.All coated magnesium disks were ultrasonically cleaned in acetone so as to remove the loose deposites.As shown in Fig.1,HA and DCPD coatings were formed on the different samples.However,the coatings characterization and formation mechanism will be investigated in details in future so that the focus of this paper can be kept on the biological work.

2.2.Magnesium samples preparation

The coated magnesium samples were immersed in 70%ethanol for 10 min,rinsed twice with distilled water,and dried under UV light.The samples were introduced into a 24-well plate(BD Falcon,BD Biosciences,Broendby,Denmark),with 5×104primary HTCF cells per well(Invitrogen CountessTM).In vitroexperiments,titanium was used as a positive control due to its well accepted biocompatibility.And glass was set as a blank control in order to obtain natural data of cell death rate for comparing.Trabeculectomy was used as controlin vivoexperiments.

Fig.1.XRD spectra of the coated samples.

2.3.Primary cell culture

Tenon’s capsule samples were obtained from 32 glaucoma patients with no prior history of ocular surgery.The study was approved by Health Sciences Research Ethics Board of Western University and complies with the Declaration of Helsinki.All participants provided informed consent.The capsule samples were excised during surgery and cultured in a dish with 60mm diameter.The culture medium,consisting of Dulbecco’s Modified Eagle’s medium(DMEM,Gbico,USA)supplemented with 10% fetal bovine serum(FBS,Gbico,USA)and 1% penicilin/streptomycin(Gbico,USA),was changed every 2–3 days and the cells were allowed to reach 80% confluence.Subsequently,the cells were disaggregated with 0.25% trypsin and 0.02% EDTA at 37°C for 5min,transferred to 25 cm2flasks(BD Falcon,BD Biosciences,Broendby,Denmark),and placed in a 37°C 5%CO2incubator for subculture.The cells were seeded in a 24-well plate when they reached 80% confluence.

2.4.Assessment of morphology

The cells were analyzed 2 days after seeding.Cell counting was performed by fluorescence microscopy(Axio Observer.Z1;Carl Zeiss,Germany)using Heochst dye(Molecular Probes,Carlsbad,CA,USA)to visualize the nuclei.After 10 min of incubation with the fluorescent dye,the cells were fixed with 4% buffered formaldehyde in phosphate buffered saline(PBS)for 10 min,and then permeabilized with 0.1%Triton-X 100 in PBS for 5 min.The samples were rinsed with PBS,stained with a 1:100 dilution of monoclonal mouse antivimentin and 1:500 monoclonal rabbit anti-keratin,and left overnight at 9°C.Following a PBS rinse,the samples were incubated in a 1:625 dilution of anti-mouse Alexa Fluor 488-conjugated goat anti-mouse IgG for 1 h at room temperature.For double labeling,the samples were also incubated in a 1:500 dilution of Alexa Fluor 568-conjugated donkey antirabbit IgG for 1 hour at room temperature.Lastly,the samples were rinsed twice with PBS and kept in PBS at 4°C until observation.Image sampling,cellular morphology assessment and cell counting were performed using custom-written routines for the AxioVision rel.4.7 software(Carl Zeiss).

2.5.Scanning electron microscope(SEM)observation

The primary HTCFs were seeded(5×104cells per well)on glass,titanium,and three coated magnesium disks in a 24-well culture plate and placed in a 37°C 5%CO2incubator for 24 h.The samples were rinsed with ice-cold PBS three times,fixed with 1ml of 3% glutaraldehyde and placed in 4°C for 12 h.After that,they were rinsed with 0.9% saline twice(5min per times),dehydrated by ethanol from low to high concentration(30%,50%,70%,85% and 90%,respectively)and dehydrated by 100% ethanol twice.Before observed using a SEM(S-3400N II,Hitachi,Japan),the samples were replaced by tertiary-butanol from low to high concentration(30%,50%,70%,respectively,5min per times)and replaced by 100% tertiary-butanol twice(5min per times).

2.6.MTT assay

The MTT assay assesses cellular metabolic activity.The primary HTCFs were seeded on DCPD,DCPD+SA,and HA coated magnesium disks in a 24-well culture plate,with 8 wells for each kind of sample and 2×104cells per well from Day 2 to Day 7.Glass and titanium disks were used as controls.The Thiazolyl Blue Tetrazolium Bromide(MTT)(Sigma-Aldrich,St.Louis,Missouri,USA)assay was performed according to the manufacturer’s instructions.Absorbances were measured at 575nm using a microplate reader(Molecular Devices,Sunnyvale,CA).Control cells,which represented 100% metabolic activity,were used to normalize absorbances.At least 6 samples per group were measured in duplicate.

2.7.Lactate dehydrogenase(LDH)assay

The supernatant of different groups was collected from Day 2 to Day 7.Glass was used as control.The collected supernatant was analyzed using an LDH cytotoxicity kit(Roche Life Science,Laval,Quebec,Canada),and the assay was performed according to the manufacturers’instructions.Optical densities of the solutions were read at dual wavelength of 490/655nm.Control cells,which represented 100% cytotoxicity,were used to normalize absorbances.At least 6 samples per group were measured in duplicate.

2.8.BrdU assay

According to the manufacturers’instructions,the proliferation of HTCFs was monitored using a 5-bromo-2-deoxyuridine(BrdU)Cell Proliferation Assay Kit(Merck KGaA,Darmstadt,Germany).The plate was read using a spectrophotometer microplate reader with dual wavelength set at 450/595nm.The glass group was used as control.At least 6 samples per group were measured in duplicate.

2.9.Protein extraction and western blot analysis

A western blot was performed on HTCFs samples derived from glaucoma patients.After treatment,the cells were rinsed with ice-cold PBS twice,and the total cell proteins were extracted using RIPA lysis buffer(20mM Tris,150mM NaCl,1 Mm EDTA,1% Triton X-100)containing phosphatase inhibitors and protease inhibitors.Protein concentrations were determined by micro BCA protein assay(Thermo Scientific,CA).Following the manufacturer’s instructions,10% SDSpolyacrylamide gel and 5% stocking gel were prepared and placed inside the electophorator containing running buffer.A 6uL marker(Invitrogen,Thermo Fisher Scientific,CA)was loaded on the left lane,followed by 10mg of each sample.The gel was run at 120V for 5% stocking gel and at 100V for 10% SDS-polyacrylamide gel.Subsequently,the gel was transferred to a polyvinylidene difluoride(PVDF)membrane(Invitrogen,Thermo Fisher Scientific,CA)using a Bio-Rad gel-blotting apparatus(Invitrogen,Thermo Fisher Scientific,CA).Membranes were blocked in 5% milk in Tris-Buffered Saline Tween(TBST;10mM Tris HCl,150mM NaCl,0.1%Tween 20)for 1 hour,and probed with antibodies againstα-smooth muscle actin(α-SMA,Abcam,USA)at a dilution of 1:1000.The membrane was then incubated with a primary antibody overnight at 4°C and with a peroxidaseconjugated secondary antibody for 1 hour at room temperature.After these incubations,membranes were washed in TBST for 30 min.A western blot detection kit(Western-Bright Quantum,USA)was used to visualize enhance chemiluminescence(Chemi genius2,Syngene,USA)and exposure.To ensure equal protein loading,nitrocellulose membranes were stripped with stripping buffer(Restore,Thermo Scientific,CA)and reprobed with anti-glyceraldehyde-3phosphate dehydrogenase(GAPDH)antibody at a dilution of 1:2000(Santa Cruz,Biotechnology,USA).At least 6 samples per group were measured in duplicate.

2.10.Rabbit model of trabeculectomy

All animal experiments were conducted according to the Chinese animal welfare legislation and the ARVO Statement for the use of Animals in Ophthalmic and Vision Research.In this study,25 New Zealand rabbits(mean weight:1769g;range:1238–2432g)were acquired from Chongqing medical university,China.The New Zealand rabbits were housed in clear plastic cages(60 cm2)on standard bedding.Water and a standard pellet diet were given ad libitum.All animals were randomly assigned to five treatment eyes and one control eye(trabeculectomy alone)of three animals each.

The rabbits were anaesthetized by intraperitoneal(i.p.)injection of 10mg/mL chloral hydrate(Sangon Biotechnology Co.,Ltd.)before the operation was conducted.All subjects underwent a standard primary trabeculectomy.A fornix-based conjunctival flap was utilized in all cases.A partial thickness scleral flap was constructed and dissected anteriorly to the limbus and undermined into clear cornea.The sclerotomy was performed by excision with scissors or by a punch followed by a peripheral iridectomy.Five different disks:one titanium disk,one pure magnesium disk and three coated magnesium disks(HA,DCPD,DCPD+SA)were placed under the scleral flap.The trabeculectomy flap was closed with 10–0 nylon sutures.The Tenon capsule and conjunctiva were closed with an 8–0 vicryl suture.Postoperatively,all eyes received tobramycin-dexamethasone,2 times/day.

2.11.Analysis of ion concentration in aqueous humor

Before the rabbits were euthanized,0.1ml of aqueous had been extracted by puncture of the anterior chamber from each eye and frozen at?20°C immediately.A chemistry analyzer(AU5800,Beckman Coulter,USA)was used to analyze the ion concentration of the aqueous.At least 6 samples per group were measured in duplicate.

2.12.Histology and immunofluorescent analysis

Rabbits were euthanized on day 60 after surgery.The eyes were enucleated,fixed in 4% paraformaldehyde and paraffin embedded.The samples were sectioned(4μm in thickness)with the Microm HM550(Carl Zeiss Ltd.,Oberkochen,Germany).The sections were washed with xylene twice for 15 min each,and then the slides were immersed in a series of ethanol solutions for 5 min each(twice in 100%(vol/vol),once in 95%,once in 80%,once in 70%),followed by 2-minute exchanges in water.The sections were stained with hematoxylin and eosin(H&E)for histology analysis.

2.13.Western blot analysis

The conjunctival tissues at the surgical sites were dissected and stored at?80°C for Western blot analysis.The conjunctival tissues were homogenized and lysed in Laemmli buffer(Bio-Rad),followed by boiling and centrifugation to obtain lysates.Protein concentrations were determined using a BCA protein assay kit.Proteins were separated on 10% SDSPAGE and electro-transferred to a nitrocellulose membrane.Western blotting was performed as previously described,using antibodies againstα-SMA and collagen I.Quantification was performed by measuring intensity of the signals using Quantity One,version 4.6.2 software(Bio-Rad).At least 6 samples per group were measured in duplicate.

2.14.Statistical analysis

The MTT and LDH assay results were analyzed using two-way ANOVA test.The BrdU assay,aqueous ion concentration and Western blot results were analyzed using oneway ANOVA test.Statistical significance was accepted atp<0.05.All analyses were performed using SPSS software(IBM Corp.Version 24.0.Armonk,NY,USA).

Fig.2.Immunostaining and characterization of primary Human Tenon’s Capsule cultures.Cells were stained for nuclei(blue),vimentin(green),and keratin(red)under 40x magnification.(A)shows a complete image while(B),(C),and(D)show isolated images for nuclei,vimentin,and keratin stains respectively.Images displayed are representative of all images of samples taken.

3.Results

3.1.Morphological observation and identification of HTCFs by immunofluorescence staining

After been cultured for 7 to 10 days,cells were migrated out of the tissue.Microscopically,the cells exhibited a fusiform appearance with a clear outline,and the cellular plasma was abundant,bright,and uniform in size.The cells were arranged in fasciculus or swirling patterns.No differences were observed between the recovered cells and those before cryopreservation in terms of morphology and growth characteristics.Immunofluoresence revealed that the cells were positive for the expression of vimentin,with the specific fluorescence observed within the cytoplasm(Fig.2).The cells stained negative for the expression of keratin,supporting their identity as fibroblasts.

3.2.SEM images of seeded HTCFs

The HTCFs seeded on glass and titanium disks performed numerous spindle or stellate-shaped patterns with multiple cell processes and regular oval nucleus(Fig.3A and B).Fewer cells were seen in DCPD(Fig.3C)and DCPD+SA(Fig.3D)groups which showed a flat spindle-shape with less cell processes.The number of cells in the HA group(Fig.3E)was higher than that in DCPD and DCPD+SA groups.The cells were smaller than the glass and titanium group,spindle and stellate shaped,unclear outline,with different lengths and thickness of cell processes.

3.3.Time and cellular metabolic activity curves

The succinic acid dehydrogenase in mitochondria of living cells react with exogenous MTT to produce purple crystalline nail(Formazan)and deposit in cells,while this reaction does not occur in dead cells.Hence the MTT assay is widely used to determine the viability or metabolic activity of cells.The multiple curves represented the variation of metabolic activities of HTCFs which were treated by different disks from day 1 to day 7(Fig.4).The p value of different treatments was statistically significant(ptreatment<0.001).The metabolic activity of the DCPD+SA group,which gradually declined from day 2 to day 7,was significantly lower(p<0.05)than that of the other groups.The HTCFs seeded on the titanium plates had the highest viability.The HA group showed a higher metabolic activity during the first two days,but the viability decreased gradually and became lower than that of the DCPD group from day 4 on.The p value of time was not statistically significant(ptime=0.195),indicating that time had no influence on the metabolic activity of HTCFs.

3.4.Time and cytotoxicity curves

Lactate dehydrogenase(LDH),abundant in the cytoplasm,will be released to extracellular when cell is damaged or dead.Therefore,the cytotoxicity of the materials to cells can be obtained by measuring the concentration of LDH in cell culture medium.The LDH assay was utilized to examine the cytotoxicity of coatings and necrosis of HTCFs seeded on different disks.Liberated nicotinamide adenine dinucleotide,formed from LDH’s conversion of lactate into pyruvate,subsequently reduced a tetrazolium dye into formazan,which could be detected using a spectrophotometer.The p value of the different treatments was statistically significant(ptreatment<0.001)(Fig.5).The HA group had the lowest cytotoxicity,which was statistically different(p<0.05)compared with other groups.The cytotoxicity of the DCPD+SA group was higher than that of the HA and DCPD groups,but similar to that of the titanium(p=0.468)and glass(p=0.976)groups.The p value of time was not statistically significant(ptime=0.260).

3.5.Cell proliferation study

EdU is a thymine nucleoside analogue which replaces thymidine to infiltrate in the replicating DNA during cell proliferation.Therefore,the proliferation of HTCFs is accurately determined by detecting EdU markers.As shown in Fig.6,the HTCFs seeded on titanium disks showed a greater proliferative ability;the cell proliferation rate of the titanium group was significantly higher than that of the glass group(p=0.049).The proliferation rate of the DCPD+SA group was significantly lower compared to the glass group(p=0.047);that of the DCPD group was almost the same as the glass group;and the HA group had a slightly higher proliferation rate than that of the DCPD group.In addition,all types of coated magnesium showed a lower proliferation rate compared to the titanium group.

Fig.3.Scanning electron microscope(SEM)images of HTCFs seeded on(A)glass,(B)titanium,(C)DCPD coated magnesium,(D)DCPD+SA coated magnesium and(E)HA coated magnesium disks.The pictures were taken under 400x(i)and 800x(ii)magnification.

Fig.4.Cellular metabolic activity of different samples.MTT results from HTCFs treated with different coated magnesium.The multiple curves represent the variation of metabolic activities of HTCFs which were treated by different disks from day 1 to day 7.The p value of different treatments was statistically significant(ptreatment<0.001).Data was analyzed using two-way ANOVA test.???ptreatment<0.001,ptime=0.195.

3.6.Western blot analysis

Theα-SMA is a protein marker of myofibroblasts(MFs)which cause fibroblast proliferation and wound scarring[25].This specific protein can be detected by antibodies through Western Blot assay.Normalized to GAPDH expression,the glass group had the highestα-SMA expression(Fig.7).

Fig.6.Relative proliferation rate of HTCFs.The cell proliferation rate of the titanium group was significantly higher(p=0.049)whereas the proliferation rate of the DCPD+SA group was significantly lower(p=0.047)compared to the glass group.Data was analyzed using one-way ANOVA test(SPSS 24).

Theα-SMA expression level of the titanium group was slightly lower than that of the glass group.The different coated magnesium samples all had lowerα-SMA expression levels than that of the titanium group or the glass group.The DCPD group and the DCPD+SA group had similarα-SMA expressions.The HA group had the lowestα-SMA expression level,which was significantly lower than that of the glass group(p=0.037)and the titanium group(p=0.001).

3.7.Clinical examination and eye response

Fig.5.Time and cytotoxicity curves of different samples.The p value of the different treatments was statistically significant(ptreatment<0.001).The HA group had the lowest cytotoxicity,which was statistically different(p<0.05)compared with other groups.The cytotoxicity of the DCPD+SA group was higher than that of the HA and DCPD groups,but similar to that of the titanium and glass.Data was analyzed using two-way ANOVA test.???ptreatment<0.001,ptime=0.260.

Following surgery,the rabbits didn’t exhibit any adverse effects.60 days later,there was no congested conjunctiva,no obvious inflammation in the anterior chamber,lens and vitreous transparency(Fig.8C),and no bleeding and pigmentation(Fig.8D)in the posterior segment of eye.

Table 1Ion concentration in the aqueous humor at 60 days post-surgery(mmol/L).

Fig.7.α-SMA protein expression normalized to GAPDH expression.The different coated magnesium samples all had lowerα-SMA expression levels than that of the titanium group or the glass group.The HA group had the lowestα-SMA expression level,which was significantly lower than that of the glass group(p=0.037)and the titanium group(p=0.001).Data was analyzed using one-way ANOVA test.

3.8.Analysis of ion concentration in aqueous humor

Different coated magnesium disks and titanium alloy disks were placed under the scleral flap during surgery.The degradation of the coated magnesium may cause ion concentration variation in the anterior chamber;therefore,the ion concentration of the aqueous humor was analyzed before the rabbits were euthanized.The concentrations of potassium,sodium and chloride in the coating groups were slightly lower than other groups,and the calcium ion concentration was slightly higher than the other groups(Table 1).The pure magnesium group had the highest magnesium ion concentration at 60 days post-surgery.No statistical significance was observed in any of the groups.

3.9.Histology

Representative H&E staining images of the specimens are shown in Fig.9.The inflammatory response to surgery with different pretreatment processes was evaluated 60 days after surgery.A typical infiltration of granulocytes and macrophages could be observed at this stage of wound healing.Varying inflammatory responses to surgery was observed with the different pretreatment processes.The varying thickness in conjunctival tissue at the surgery site provided an initial indication of the inflammatory response level.An intense inflammatory response to pure magnesium treatment was observed(Fig.9C),whereas the width of the granulation tissue in the subconjunctival space at the surgery site was thicker than that observed in other groups.Fibroblast infiltration and newly formed blood vessels could also be visualized.In contrast,fibroblast infiltration was mostly adjunct to the conjunctival epithelium in the trabeculectomy alone and HA group(Fig.9A,D).In titanium,DCPD and DCPD+SA treated eyes(Fig.9B,E,F),scattered fibroblasts and inflammatory cells were observed.The inflammatory response was more intense than trabeculectomy alone and HA group.

3.10.Western blot analysis

Western blot analysis of the tissue samples at day 60 was conducted to detect collagen-1 andα-SMA content(Fig.10).Collagen-1 andα-SMA levels in the pure magnesium group was increased 60 days after surgery and remained higher than the control group.In contrast,collagen-1 andα-SMA expression was significantly lower in the titanium,HA,DCPD and DCPD+SA groups compared to controls.The DCPD,HA and the DCPD+SA group had similarα-SMA expressions.The HA group showed the lowest level of collagen-1.

4.Discussion

4.1.Time and treatment

A prior study reported that the logarithmic phase of HTCFs was from 48 h to 7 days[26].In order to examine the relationship between the HTCFs’metabolic activity and time,and to investigate the variation of cytotoxicity during this period,the MTT and LDH assays of treated HTCFs was performed from day 2 to day 7.The results revealed that no statistically significant difference was observed in metabolic activity or necrosis at different time points during the logarithmic phase(p=0.195,p=0.260,respectively).Based on these findings,the factor of time was not a major contributor to reduced metabolic activity and increased cytotoxicity in thisin vitroexperiment.Therefore,the results describe the influence of different treatments on HTCFs during the logarithmic phase.

Fig.8.Intraoperative and postoperative photos.Five different disks were placed under the scleral flap,including one titanium,one pure magnesium and three coated magnesium disks(HA,DCPD,DCPD+SA).(A)A rabbit underwent a standard primary trabeculectomy.(B)The subjects were inspected regularly under slit lamps postoperatively.(C)The anterior segment of the eye 60 days post-surgery.(D)The posterior segment of the eye 60 days post-surgery.

4.2.Biocompatibility of coated pure magnesium

The purpose of this study was to evaluate the biocompatibility of coated pure magnesium which could be promising materials for novel drainage devices.Lorenz A et al.[27]reported that cell death on untreated pure magnesium samples occurred within 1 day,whereas surface passivation could enable survival of a number of cells on magnesium.However,in this work,cell densities were reduced on magnesium samples even with prior passivation treatments,compared with glass substrates used as a reference.Cell death was thought to be related to the ongoing corrosion of magnesium in cell culture medium,leading to a pH increase.A recent study indicated that cell culture tests could not provide enough volume to compensate for the high concentration of Mg2+ions or the alkaline pH shift in solution,and hence might not be appropriate for testing resorbable materials[28].Although coatings could reduce Mg2+release from pure magnesium matrix and prevent pH increase,the coatings might influence the viability of cells and their cytotoxicity might lead to necrosis of HTCFs.

In the MTT and LDH assays,the p value of different treatments was statistically significant(p<0.001).The DCPD+SA group had the lowest metabolic activity but higher cytotoxicity than the other two coatings.Both the HA and DCPD coatings had lower cytotoxicity than titanium and glass.

HA is attributed to the compositions of bioactive and bioresorbable ceramics which form HA crystals by reacting with the organism at the implant-biomedium interface[29].Synthetic HA is a complete chemical and crystalochemical analog of bone mineral.This chemical similarity with bone accounts for their osteoconductive potential and excellent biocompatibility[30,31].In fact,HA,as a biological material,has been used in ophthalmological clinical practice for a long time,such as HA orbital implants.Due to its excellent biocompatibility,HA orbital implant exposure rarely causes rejection after implantation.

Fig.9.Hematoxylin and eosin staining of trabeculectomy sections.Sections of(A)trabeculectomy alone,and(B)titanium,(C)pure magnesium,(D)HA,(E)DCPD and(F)DCPD+SA treated eyes at 60 days post-surgery.

4.3.Coated magnesium attenuate HTCFs proliferation

Studies have shown that fibroblasts play significant roles in wound repair,contraction,and scar formation[32,33].In quiescent stage,fibroblasts have small cell bodies and long spindle shape,the rough endoplasmic reticulum and golgi complex of fibroblasts are underdeveloped.However,the proliferation function could be activated when fibroblasts are stimulated,such as surgery or trauma.Therefore,inhibiting the proliferation of fibroblasts is crucial to regulate postoperative scarring.

According to the BrdU cell proliferation assay,coated magnesium samples were associated with lower proliferation rates of HTCFs compared to titanium.In the BrdU assay,the same number of cells was loaded to each well at the beginning,but due to the lower cytotoxicity and better metabolic activity of cells seeded on the titanium disks,more cells grew and proliferated on the titanium disks 48 h later.The DCPD+SA coating group had higher cytotoxicity and lower metabolic activity compared to the other coatings,thus a lower proliferation rate.On the other hand,images from the SEM showed HTCFs had difficulty attaching on the DCPD and DCPD+SA coating due to the uneven surface.Additionally,the corrosion of the pure magnesium matrix and coating,the complex deposits and the change of pH might further inhibit the attachment and proliferation of HTCFs.

The inert materials with better biocompatibility,such as titanium alloys,are more likely to result in fibroblasts proliferation and to cause scarring.Therefore,it is desirable to develop a novel glaucoma surgical device with scarring-prevention properties based on the biodegradable coated magnesium.

4.4.HA coating inhibitsα-SMA expression

HTCFs are undifferentiated mesenchymal cells that exist in subconjunctival connective tissue.Stimulated by surgery,HTCFs transform to MFs which are the expression ofα-SMA[34].MFs response for contracture of wounds,and have some characteristics of smooth muscle,such as contractile properties and fibrils.They are also believed to produce collagen and other extracellular matrix,the cause of postoperative wound fibrosis.

As mentioned previously,HA orbital implants have been widely used in clinical practice with excellent performance,and no obvious adverse effects have been reported[35].The results of from the western blot are more reliable and convincing as the data ofα-SMA expression had been normalized to that of GAPDH.The expression of theα-SMA protein is a marker of myofibroblasts,which reflects the number and proliferation of myofibroblasts[36].Also,it is an important marker to estimate scar formation and surgical prognosis.In our experiment,theα-SMA expression levels decreased in all of the coated magnesium groups.Furthermore,when compared to glass and titanium,the HA coating reduced the expression ofα-SMA significantly.As seenin vivo,the observations suggest that collagen deposition might be altered in HA-coated Mg-treated eyes.The survival of the bleb is partially due to reduced deposition of collagen fibers andα-SMA–positive myofibroblasts,which lead to deficient maturation of the scar at the wound site.

Fig.10.Western blot analysis ofα-SMA and collagen-1 protein expression normalized toβ-actin expression.Collagen-1 andα-SMA expression was significantly lower(p<0.001)in the titanium,HA,DCPD and DCPD+SA groups compared with controls.The DCPDHA and the DCPD+SA group had similar α-SMA expressions.The HA group showed the lowest level of collagen-1.Data was analyzed using one-way ANOVA test.

5.Conclusions

In this paper the biocompatibility and anti-scarring potential of magnesium coated with HA,DCPD and DCPD+SA were investigatedin vitroandin vivo.The results show that coatings are able to affect the interactions of magnesium with cells and tissues.Among the three samples,HA coated magnesium has the lowest cytotoxicity and excellent biocompatibilityin vitro,which can inhibitα-SMA protein expression and prevent scarring.

As shown by the implantation experiments,the rabbits didn’t exhibit any adverse effects 60 days later post-surgery.Slit-lamp examination showed no congested conjunctiva,no obvious inflammation in the anterior chamber,lens and vitreous transparency,and any bleeding and pigmentation.The ion concentrations of the aqueous humor showed no statistical significance in any of the coated groups as compared with the titanium and trabeculectomy controls.

Although an intense inflammatory response to pure magnesium treatment is observed,the inflammatory response of HA coated magnesium is similar to that of trabeculectomy alone.Therefore,HA coated magnesium may be a very promising biodegradable material for the next generation of glaucoma drainage devices.

Declaration of Competing Interest

The authors declare no conflict of interest.

Acknowledgments

Thanks to the secretaries and staff of Daping Hospital,Joseph’s Hospital,The Third Affiliated Hospital of Chongqing Medical University and National Engineering Research Center for Magnesium Alloys where this research was conducted.This research was funded by Natural Science Foundation of China(Grant number 81470629),Natural Science Foundation of Chongqing(Grant number cstc2018jcyjAX0016).