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Biomaterials 30 (2009) 3143–3149

Contents lists avai

Biomaterials

journal homepage: www.elsevier .com/locate/biomateria ls

The in vitro corneal biocompatibility of hydroxyapatite coated carbon mesh

Susan R. Sandeman*, Hannah Jeffery, Carol A. Howell, Martin Smith, Sergey V. Mikhalovsky,Andrew W. LloydBiomedical Materials Research Group, School of Pharmacy and Biomolecular Sciences, Cockcroft Building, University of Brighton, Brighton, East Sussex BN2 4GJ, UK

a r t i c l e i n f o

Article history:Received 19 December 2008Accepted 28 February 2009Available online 19 March 2009

Keywords:CarbonBiocompatibilityCorneaCytokineWound healing

* Corresponding author. Tel.: þ44 01273 642015; faE-mail address: s.sandeman@brighton.ac.uk (S.R. S

0142-9612/$ – see front matter Crown Copyright � 2doi:10.1016/j.biomaterials.2009.02.042

a b s t r a c t

The purpose of this study was to consider the use of a hydroxyapatite (HA) coated porous carbon matrixas a synthetic dental laminate substitute in osteo-odonto-keratoprosthetic (OOKP) design. 3 types ofcarbon meshes were coated with HA by sonoelectrochemical deposition. The materials were charac-terised by scanning electron microscopy (SEM) and HA deposition was characterised by elementalanalysis and X-ray diffractometry (XRD). In vitro assays were carried out to quantify the effects of HAcoating on human keratocyte adhesion. Cellular cytokine production was used to assess inflammatorypotential. HA coating significantly increased keratocyte adhesion to the carbon matrix (p< 0.01). Thematerials did not induce excessive cytokine production by the adherent keratocytes. In addition, thematrices themselves adsorbed significant levels of the cytokine IL-8 (p< 0.05). The results indicate thatHA coated carbon matrices provide a suitable environment to enhance in-growth of corneal cells withoutinducing further inflammation. The materials may also suppress excessive inflammation by adsorption ofthe cytokine IL-8 into the porous, internal carbon structure.

Crown Copyright � 2009 Published by Elsevier Ltd. All rights reserved.

1. Introduction

Corneal opacity, as a result of injury or disease, is the secondmost common cause of blindness globally, affecting over 10 millionpeople worldwide [1]. While corneal transplants have a successrate of over 80% factors such as allograft rejection, donor availabilityand a shrinking donor pool have lead to increased interest inalternatives to donor transplantation [2].

Various Keratoprosthetic devices (KPros) have been designed totreat bilateral, corneal blindness where corneal grafts repeated fail[3,4]. These consist of a central optic which allows light to enter theeye and a peripheral skirt designed to promote implant retention.The majority of designs have not achieved long-term clinicalsuccess with the rejection of the implant by the surrounding,inflamed ocular tissue over time. The devices which have shownmost potential are those with the combined properties of opti-mised design, ocular biocompatibility, corneal structural mimicry,or with a natural, autologous component such as the dental lami-nate skirt of the osteo-odonto-keratoprosthesis (OOKP) [5–9]. Thelong-term success of the OOKPs, particularly in dry eyes, is due inpart to the stability, porosity and hydroxyapatite (HA) rich envi-ronment created by the dental lamina for corneal integration and tothe buccal epithelial barrier to infection across the corneal surface.

x: þ44 01273 642674.andeman).

009 Published by Elsevier Ltd. All

However, despite its success there are still a great number ofcomplications associated with OOKP usage including laminateresorption, surgical complexity and the absence of autologousmaterial [10].

The following in vitro study investigated the use of a hydroxy-apatite coated, activated carbon matrix as a synthetic substituteskirt material. The biologically inert nature of the carbon itselflimits the inflammatory response and material resorption. Inaddition, the highly porous structure of the carbon mesh can becontrolled to adsorb inflammatory cytokines and limit excessiveinflammation in the surrounding tissue [11]. HA coating is alsoknown to enhance cell integration in orthopaedic and ophthalmicapplications [12,13]. The study utilised a sonoelectrochemicaldeposition technique to produce highly adhesive HA deposits onthe carbon surface [14]. The purpose of the study was to confirmconsistent HA deposition, consider the effect of HA deposition onkeratocyte adhesion, consider the production of cytokines byadherent keratocytes as a measure of corneal biocompatibility andto consider the adsorptive capacity of the carbon mesh forinflammatory cytokines IL-6 and IL-8.

2. Materials and methods

2.1. HA coating of carbon meshes by sonoelectrochemical deposition

Three activated carbon mesh variants were supplied by Mast Carbon Interna-tional (M1, MU25 and M62). Replicates of each carbon mesh type were coated bysonoelectrodeposition according to the method of Han et al. [14]. In brief, an

rights reserved.

Fig. 1. Scanning electron microscopy was used to characterise (a) carbon mesh M1structure and (b) hydroxyapatite (HA) deposition onto the carbon fibres (mag. �500).

S.R. Sandeman et al. / Biomaterials 30 (2009) 3143–31493144

electrolyte solution consisting of 9.9 mM calcium nitrate, 5.9 mM ammonium phos-phate buffered to pH 5.5 with 3.5% ammonium hydroxide was heated to 50 �C in anultrasonic water bath set at 60 kHz. A carbon mesh cathode was submerged in thewater bath 1 cm apart from a platinum electrode and a constant current of 20 mAwas applied over 45 min for HA crystal deposition. Coated meshes of each type wereprepared then rinsed in sterile dH2O and dried at 24 �C.

2.2. Characterisation of HA coating by SEM and XRD

A qualitative study of carbon mesh macroporosity and deposited crystalmorphology was carried out using a JEOL JSM-6310 Scanning electron microscope(SEM) set at 10 kV. Samples were mounted onto aluminium stubs and sputter coatedwith palladium using a positron SC7640 sputter coater prior to analysis. EnergyDispersive X-ray microanalysis (EDAX) was used to identify the elemental compo-sition of deposits on the mesh surface.

A Panalytical X’Pert Pro X-Ray Diffractometer with copper Ka radiation set ata voltage of 40 kV and a current of 40 mA was used to analyse the form of calciumphosphate deposited onto the carbon meshes. A 10 mm irradiated length anda spinning stage were used. Samples were loaded directly onto a sample holder ascarbon meshes. X-rays were detected from 5 to 70� 2q using 0.008� steps, resultingin a 20 min scan time. No evidence was found for a preferred crystallographicorientation.

2.3. Measuring the effect of hydroxyapatite coating on keratocyte adhesion andinflammatory cytokine production

An LR99 human keratocyte cell strain established by the Biomedical MaterialsResearch Group at the University of Brighton was used for the in vitro cell adhesionexperiments [15]. Cells were cultured in DMEM containing 10% Foetal calf serum and

1% penicillin/streptomycin and passaged by standard trypsinisation. Four samples ofeach type of HA coated and uncoated carbon mesh were sterilised under UV light in24 well plates and pre-conditioned in supplemented cell media. 5�104 cells in0.5 ml media were seeded onto each disc. A negative control of cells on tissue cultureplastic (tcp) and a positive control of cells on tcp spiked with 10 ng/ml lipopoly-saccharide (LPS) was also included. The plates were incubated at 37 �C for 5 h.Conditioned media was removed and used to measure cellular production ofinflammatory cytokines IL-6 and IL-8 by ELISA according to manufacturer’sinstructions using a 1:50 dilution of sample in assay diluent (BD Biosciences). Thecells were fixed in 2.5% glutaraldehyde and cell adhesion was quantified using aninverted fluorescent confocal microscope (Leica TCS SP5). Cells were stained withphalloidin with tetramethylrhodamine and inverted onto DAPI mountant. Bluefluorescence was counted in 15 fields at �200 magnification. Live cell adhesion wasvisualised on the M1 material using a calcein-acetoxymethyl ester stain of non-fixedcells. Following confocal analysis the samples were rinsed in PBS, dehydrated andsputter coated for SEM analysis of cell adhesion to the coated and uncoated meshes.Differences in cell adhesion were tested for significance using Student’s t-test.

2.4. Carbon mesh adsorption of inflammatory cytokines IL-6 and IL-8

Each carbon mesh type was incubated in serum spiked with 400 pg ml�1 ofrecombinant human cytokines IL-6 and IL-8 at 37 �C for 24 h on an orbital shaker.The conditioned serum was removed and tested for cytokine removal by the carbonmeshes using ELISA.

3. Results

3.1. SEM, EDAX and XRD characterisation of HA coating on carbonmeshes

SEM analysis of the HA coated carbon meshes revealed an evencoating of predominantly needle-shaped crystals less than 1 mm indiameter across the surface of the mesh fibres (Fig. 1). Plate-like, flatdiscs of approximately 1 mm diameter were also observed on someof the fibres for each carbon mesh type. The carbon meshesthemselves were made up of woven fibrillar bundles. M1 and M62consisted of 200 mm width carbon fibre bundles. Each fibre withinthe bundle had a width of 10 mm. MU25 was a more finely wovenmesh and consisted of 100 mm width carbon fibre bundles witha thicker 30 mm individual fibre width. The materials had a variableintrafibrillar distance of 10–40 mm. The interfibrillar distance forMU25 was not greater than 25 mm but was as great as 100 mm forM1 and M62. EDAX analysis revealed calcium and phosphate peaksconsistent with apatite deposition (data not shown). XRD analysisproduced similar spectrums for each of the HA coated carbon meshtypes and for different repeats of the same mesh type indicatingthat the sonoelectrodeposition technique produces a consistentpattern of calcium phosphate deposition (Fig. 2a). XRD analysisrevealed that the calcium phosphate deposited was most analogouswith the known spectrum for HA rather than other calcium phos-phates. The sample peaks tended to have lower peak intensity andlarger peak widths than the standard HA peaks (Fig. 2b). MU25showed a shift in peak at 26� and a reduction in peak intensityconsistent with the presence of a small amount of monetite(Ca(HPO4)) and/or whitlockite (Ca3(PO4)2) in addition to HA.

3.2. Keratocyte adhesion and cytokine production by adherent cells

Live cell adhesion was observed on both the coated anduncoated M1 carbon meshes. The cells showed normal fibroblast,spindle-shaped morphology and were visible throughout themeshes (Fig. 3a). Keratocytes grown on the uncoated carbonmeshes were less clearly visible on the mesh surface and showedless characteristic morphology (Fig. 3b). Quantification of celladhesion indicated that HA coating significantly increased celladhesion to the carbon meshes, particularly for MU25 (p< 0.01)(Fig. 4). SEM analysis of adherent cells showed the cells’ spanning10–40 mm across the coated M1 carbon fibres (Fig. 5).

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Fig. 2. X-ray diffraction was used to characterise the consistency and type of apatite deposition onto the carbon fibres. (a) A consistent XRD pattern was obtained for the M1 samplerepeats indicating consistent apatite deposition (n¼ 3). The stick patterns indicate peak positions and relative intensities for pure hydroxyapatite (HA). (b) Some variation in XRDpatterns indicates that nucleation of HA was not uniform. Notably MU25a shows peak shifts relative to other samples and reference materials. (c) Details of diffraction peak shift atw26� showing key diffraction peaks from reference materials indicating the presence of calcium phosphate other than HA in MU25a.

Fig. 3. A qualitative, live cell adhesion study was carried out using the live cell stain calcein-acetoxymethyl ester. Cells were visualised by confocal microscopy (�200). (a) Ker-atocytes grown on the HA coated carbon mesh M1 showed characteristic, elongated and spread fibroblast type morphology; (b) Keratocytes grown on the uncoated carbon meshM1 were less clearly visible on the mesh surface and showed less characteristic morphology.

S.R. Sandeman et al. / Biomaterials 30 (2009) 3143–31493146

Conditioned media removed from the cells on the materialsafter 5 h incubation were found to contain both IL-6 and IL-8(Fig. 6). IL-8 levels were higher in each case since keratocytes beginto secrete IL-8 at an earlier time point (unpublished data). Asexpected the LPS stimulated positive controls contained muchhigher levels of both cytokines. The cytokine levels in conditionedmedia from HA coated and uncoated M1 and M62 carbon mesheswere similar to the negative control, cell only cytokine levels. Thecytokine levels in conditioned media from the HA coated anduncoated MU25 carbon meshes were significantly reduced whencompared to the negative control, particularly for IL-8 (p< 0.05).

3.3. Carbon mesh adsorption of cytokines IL-8 and IL-6

All three of the carbon meshes significantly adsorbed IL-8(Fig. 7). This was most significant for MU25 which removed 48% ofthe IL-8 present in the serum (p< 0.01). M62 adsorbed 20% of the

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Fig. 4. Keratocyte adhesion to the 3 types of HA coated and uncoated carbon meshes MU25, Min 15 fields was counted by fluorescent microscopy at �200 (n¼ 3, mean� SEM).

IL-8 and M1 adsorbed 14% of the IL-8. No significant removal of IL-6occurred for any of the carbon meshes (data not shown).

4. Discussion

This body of work has shown that sonoelectrochemical depo-sition can be used to deposit calcium phosphate, predominantly inthe form of HA onto carbon meshes. The resulting materialsencouraged keratocyte adhesion without provoking excessive pro-inflammatory IL-6 and IL-8 cytokine production. Additionally, thecarbon meshes adsorbed significant levels of IL-8, particularly inthe case of MU25.

HA is the form of calcium phosphate most closely related to themineral found naturally in calcified tissues such as bone and teeth[16]. HA coatings have been used to promote rapid fixation andbone integration of orthopaedic and dental implants since the1980s with considerable success. However, HA coating qualityremains a continuing concern in the pursuit of consistent, long-

M1 HA M62 M62aterial

0 µm100

1 and M62 was quantified using a DAPI nuclear stain of fixed, adherent cells. Adhesion

Fig. 5. SEM of keratocytes adherent to HA coated M1 carbon matrix (mag. �1500).

S.R. Sandeman et al. / Biomaterials 30 (2009) 3143–3149 3147

term implant stability [17]. Bone-derived HA orbital implants areused in ophthalmology with few of the negative side effects asso-ciated with plastic synthetics [18]. A study comparing HA discs withpHEMA and PTFE based KPro materials currently under clinical trialindicated significantly better keratocyte integration with the HAbased materials [12]. While HA based materials are a logicalsubstitute for the OOKP supporting dental laminate, resorptionstudies of coralline and commercial bone replacement materialsindicate that HA alone is too unstable to overcome currentcomplications with bone resorption of the OOKP skirt. However,a design which combines a biointegrative HA coating with thestability of an underlying, inert, carbon fibrillar frame which is notsusceptible to degradation may incorporate the benefits of bothmaterials in addition to providing a more stable long-termindwelling device.

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Fig. 6. Measurement of cytokines IL-8 and IL-6 produced by keratocytes in contact with carcytokine production by cells in contact with materials (n¼ 3, mean� SEM).

The development of an HA coating method which allowsconsistent, strongly adhesive HA deposition onto carbon is currentlyunder investigation. Previous work by Han et al. [14] developed anddemonstrated the benefits of using a sonoelectrochemical techniqueinstead of standard electrochemical methods for HA deposition ontocarbon materials. The use of ultrasound produced a more uniform,fine, needle-like crystal deposition with improved coating adhesionto the carbon surface. FTIR spectroscopy indicated that the appli-cation of untrasound encouraged the formation of PO4

3� instead ofHPO4

2� ions consistent with a reduction in the formation of otherphases of calcium phosphate such as brushite (Ca(HPO4)$2H2O) andmonetite (Ca(HPO4)) and the rising dominance of HA depositionwith this technique. The current study used XRD to confirm thepresence of calcium phosphate predominantly in the form of HACa5(PO4)3(OH). The XRD spectrums of the coated carbon meshesmost closely matched those of the known HA spectrum. The pres-ence of some flat-plate crystalline deposits, observed under SEMmay have produced the wider width peaks and lower peak intensityobserved. Peak differences at 26� particularly for HA coated MU25carbon meshes suggest the presence of some monetite and whit-lockite but predominantly HA deposition was confirmed.

While all of the carbon mesh types showed an improvement inkeratocyte adhesion following HA coating, adhesion to carbonmesh type MU25 was most significantly improved (p< 0.01). HA isthought to improve cell adhesion by the adsorption of extracellularcomponents such as fibronectin in a configuration which maxi-mises exposure of integrin binding sites and allows increased cellbinding via cell surface integrin receptors [6]. While cell adhesionmechanisms were not the focus of the current study previousstudies have shown that increased integrin receptor expressioncoincided with increased adhesion of keratocytes to hydroxyapatite[12]. The reasons why keratocyte adhesion to MU25 was mostimproved by HA deposition are not fully understood. Cells areknown to respond to surface topography and adhesion will begreatest where interfibrillar spacing does not exceed cellular span.SEM imaging revealed a broader individual fibre diameter andsmaller interfibrillar spacing on the non-coated MU25 giving

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bon meshes and HA coated carbon meshes MU25, M1 and M62 indicated low levels of

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Fig. 7. Measurement of IL-8 adsorption from human serum by the carbon meshes indicated that MU25 removed 50% of the total IL-8 in solution. M1 removed 14% and M62removed 20% of the positive control levels of IL-8 (n¼ 3, mean� SEM).

S.R. Sandeman et al. / Biomaterials 30 (2009) 3143–31493148

a smoother surface for surface cell adhesion and spreading and thehigher count shown in Fig. 4. The differences in fibrillar diameterand orientation provide a more finely woven surface for HAdeposition and probably resulted in a more exposed and regularsurface for improved cell adhesion on the coated MU25 mesh. Inaddition, the cytokine IL-8 is known to be chemotactic and itspreferential adsorption to MU25 may also have influenced kera-tocyte propensity to adhere.

The expression of cytokines IL-6 and IL-8 by keratocytes inter-acting with the materials was used as an indication of the inflam-matory potential of the carbon meshes and HA coated carbonmeshes. IL-6 and IL-8 are expressed by cultured keratocytes and areknown to moderate the corneal response to inflammation andinfection [19,20]. The secretion of IL-6 and IL-8 by keratocytes incontact with the HA coated and uncoated carbon meshes was lessthan that of cells in contact with tissue culture plastic. Neither thecarbon meshes themselves or the deposited HA induced additionalcytokine secretion by the keratocytes indicating the non-inflam-matory nature of the carbons themselves and the deposited HA.Levels of IL-8 were significantly lower for the MU25 samples. Theselevels are explained by the capacity of the MU25 carbon mesh itselfto adsorb the cytokine IL-8, removing it from the cell conditionedmedia prior to sample removal (Fig. 7). It has previously beendemonstrated that cytokine removal is controlled by the presenceof mesopores in the range of 2–50 nm within the carbon matrix.The carbon meshes used in the current study are purely micropo-rous and it is likely that the MU25 samples have micropores of lessthan 2 nm but close to the mesoporous range. The smaller 8 kDacytokine IL-8 is adsorbed within the microporous matrix but thelarge 26 kDa cytokine IL-6 is not. This is a phenomenon previouslyobserved in microporous carbon beads [21]. The use of a KPro skirtmaterial which does not exacerbate the inflamed cornea andadditionally is capable of removing inflammatory cytokines whichdrive chronic corneal inflammation is an interesting application forthe carbon matrix itself.

5. Conclusions

A sonoelectrochemical deposition technique has been used tocoat 3 types of carbon meshes with HA with potential application asan OOKP synthetic skirt substitute. The carbon meshes and HAcoating did not provoke excessive cytokine production by

keratocytes. The HA coating significantly improved keratocyteadhesion to the carbon meshes and the carbon mesh MU25adsorbed the inflammatory cytokine IL-8. The use of an HA coated,carbon mesh with the underlying capacity to remove the inflam-matory mediator IL-8 adds a novel aspect to this work with theencouragement of keratocyte integration and the potentialsuppression of corneal inflammation. Further work will considerthe stability of HA deposition and optimisation of carbon meshstructural and anti-inflammatory properties.

Acknowledgements

The authors wish to acknowledge Professor Stephen Tennison atMast Carbon International for supplying the carbon materials usedin the project and Prof. Hongmei Han for providing the HA depo-sition technique.

Appendix

Figures with essential colour discrimination. Certain figures inthis article, in particular parts of Figs. 2–4 may be difficult tointerpret in black and white. The full colour images can be found inthe on-line version, at doi:10.1016/j.biomaterials.2009.02.042.

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