Research Article A Crosslinked HA-Based Hydrogel...

Hindawi Publishing CorporationInternational Journal of BiomaterialsVolume 2013 Article ID 460437 8 pageshttpdxdoiorg1011552013460437

Research ArticleA Crosslinked HA-Based Hydrogel Ameliorates Dry EyeSymptoms in Dogs

David L Williams1 and Brenda K Mann23

1 Department of Veterinary Medicine Madingley Road Cambridge CB3 0ES UK2 SentrX Animal Care Inc 391 Chipeta Way Suite G Salt Lake City UT 84108 USA3Department of Bioengineering University of Utah 72 S Central Campus Drive Rm 2750 Salt Lake City UT 84112 USA

Correspondence should be addressed to David L Williams dlw33camacuk

Received 4 December 2012 Revised 25 May 2013 Accepted 26 May 2013

Academic Editor Bruce Milthorpe

Copyright copy 2013 D L Williams and B K Mann This is an open access article distributed under the Creative CommonsAttribution License which permits unrestricted use distribution and reproduction in any medium provided the original work isproperly cited

Keratoconjunctivitis sicca commonly referred to as dry eye or KCS can affect both humans and dogs The standard of care intreating KCS typically includes daily administration of eye drops to either stimulate tear production or to hydrate and lubricatethe corneal surface Lubricating eye drops are often applied four to six times daily for the life of the patient In order to reduce thisdosing regimen yet still provides sufficient hydration and lubricationwe have developed a crosslinked hydrogel based on amodifiedthiolated hyaluronic acid (HA) xCMHA-S This xCMHA-S gel was found to have different viscosity and rheologic behavior thansolutions of noncrosslinked HA The gel was also able to increase tear breakup time in rabbits indicating a stabilization of thetear film Further in a preliminary clinical study of dogs with KCS the gel significantly reduced the symptoms associated with KCSwithin two weeks while only being applied twice dailyThe reduction of symptoms combined with the low dosing regimen indicatesthat this gel may lead to both improved patient health and owner compliance in applying the treatment

1 Introduction

Keratoconjunctivitis sicca (KCS) commonly referred to asdry eye syndrome is an ophthalmic disorder common inhumans and dogs The reported incidence of KCS in humansvaries from 5 to 33 depending on the report and how thedata were obtained while the incidence in dogs is approxi-mately 1ndash4 [1] In general KCS results from a dysfunction ina component of the lacrimal functional unit leading tochanges in the volume composition or clearance of the tearfilm [2] The lacrimal functional unit is composed of thelacrimal glands (both main and accessory) the ocular sur-face and the interconnecting innervations [3] In dogs themost common cause of KCS is immune-related lacrimalgland disease [4 5] Other causes include congenital aplasiaof the gland drug-induced or traumatic injury to the glandand neurologic dysfunction affecting the gland [4 5] Withimmune-mediated KCS in dogs there is a predispositionfor specific breeds having a higher prevalence These breedsinclude English Bulldogs West Highland White Terriers

Cavalier King Charles Spaniels American and EnglishCocker Spaniels and Pugs with the prevalence reaching ashigh as 20 in these breeds [4 5]

There is little information about the progression of dryeye symptoms in dogs with KCS as they age but a graduallowering of Schirmer tear test values in normal dogs haspreviously been shown as they age [6] Although KCS is notconsidered a life-threatening disease it often leads to cornealdamage and scarring in the dog with concurrent increasedvascularization hyperpigmentation and vision loss To treatKCS surgery is sometimes required to transpose the parotidsalivary duct into the ventral conjunctival sac allowing salivato act as a tear replacementHowever KCS ismore commonlytreated by administering topical medications and hydratingdrops daily for the life of the animal Two common topicalsare cyclosporine acting as a lacrimomimetic to stimulateincreased tear production and a tear supplement to providemoisture and lubrication [4 7] Cyclosporine drops or oint-ment is often administered twice daily although for difficultcases the dosing regimen may be more frequent [4] The tear

2 International Journal of Biomaterials

supplement on the other hand is often administered fourtimes daily or more

Tear supplements often contain a compound to increasethe viscosity of the solution such as polyvinyl alcoholhydroxypropylmethylcellulose carboxymethylcellulose pol-yethylene glycol or hyaluronic acid (HA) [4 7 8] HA isparticularly attractive as it is a naturally occurring polysac-charide found throughout the body during all stages ofdevelopment [9 10] In the eye HA is in the aqueous humorand vitreous and also coats the corneal endothelium [11]In tear supplements the viscoelasticity of HA leads to areduction of tear removal and an increase in tear stabilitythereby reducing some of the symptoms of dry eye [12 13]

Due to this viscoelasticity one means of characterizingHA-based products is to determine the rheologic propertiesof the eye drop or gelThese properties are important to assesssince the molecular weight and concentration of the HA willaffect the viscosity as well as elastic and viscous shear moduliof the solution [14 15] Additionally the concentration ofions such as salts in the solutionmay influence the rheologicproperties due to the polyanionic nature of HA [16 17]Several HA-based products with varying rheologic propertieshave been developed for ophthalmic surgery [18 19] andthese properties may influence comfort and efficacy in a dryeye formulation [14]

Although HA-based tear supplements have been used forover 20 years a formulation that extends the contact time ofthe HA with the ocular surface may allow for less frequentapplication reducing the overall cost and burden on thepatient and in the case of dogs the owner One method ofextending the contact timemay be to covalently crosslink theHA rather than the physical or ionic crosslinking that occursin solutions of high molecular weight HA A modified HAthiolated carboxymethyl HA (CMHA-S) has previously beenused to create crosslinked hydrogels for treating skin andcorneal wounds in multiple species [20 21] Here we havedeveloped a new hydrogel formulation as a tear supplementfor treating KCS in dogs The hydrogel was characterizedusing rheology to compare to noncrosslinked solutions ofHA The crosslinked CMHA-S hydrogel was then used ina clinical setting to treat dogs previously diagnosed withKCS monitoring response to therapy by evaluating tearproduction (Schirmer tear test) conjunctival hyperaemiaocular discharge and ocular irritation as determined byblink frequency and palpebral aperture narrowing With thisnew crosslinked hydrogel formulation a reduction in bothrequired application frequency and in KCS symptoms isanticipated and should improve both patient corneal healthand owner compliance

2 Materials and Methods

21 Crosslinked CMHA-S Hydrogel CMHA-S was syn-thesized as previously described [21] Thiol modification(3 times 10minus4mmolmg) was assessed using 551015840-dithio-bis(2-nitrobenzoic acid) (Ellmanrsquos reagent Sigma-Aldrich) MW(340 kDa) was assessed using gel permeation chromatog-raphy and dynamic light scattering Purified CMHA-S was

diluted to a final concentration of 4mgmL in phosphate-buffered saline (PBS pH 74 Fisher Scientific) The CMHA-Ssolution was then sterilized through a 02120583m filter intoa sterile mixing bowl The solution was crosslinked usinga dilute solution of sodium hypochlorite (Sigma-Aldrich)while mixing and the resultant gel was mixed overnightThe crosslinkedCMHA-S gel (xCMHA-S) was then packagedaseptically into sterile 15mL eye drop bottles A solution ofnoncrosslinked HA (900 kDa Novozymes) at approximately4mgmL in PBS (hereinafter referred to as HA4) was madefor comparison in the rheological studies below

To verify the final concentration of CMHA-S in the pack-aged xCMHA-S gel and HA in the noncrosslinked solution(HA4) a carbazole assay was used which detects uronic acid[22]TheHA concentration in a commercial eye product thatis a noncrosslinked solution of HA (Clinadry Eye LubricantIDPHAR Belgian Pharmaceuticals) (hereinafter referred toas HA2) was also assessed using this assay Briefly 250 120583Lof sample or standard was combined with 15mL ice-cold0025M sodium tetraborate decahydrate (Sigma-Aldrich) inconcentrated sulfuric acid (Acros Organics) and then heatedin boiling water for 10min followed by rapid cooling in an icebath 50120583L of 0125 carbazole (Sigma-Aldrich) in analyticalgrade methanol (Acros Organics) was added to each sampleand standard The mixtures were heated in boiling water for15min and then cooled rapidly to room temperature Theabsorbance at 530 nm was measured on a spectrophotometer(Genesys 10S Thermo Scientific) Six samples were used foreach formulation standards used here were solutions of HAin PBS

22 Rheological Assessment Rheological testing was per-formed on an AR1000 rheometer (TA Instruments) using a25mm diameter parallel plate format Samples (5-6mL) wereplaced in a 35mm plastic Petri dish and the rheometer headlowered to a gap distance of 5mm To determine viscosityshear rate was varied from 01 to 100 sminus1 A strain sweep wasthen performed at an oscillation frequency of 1Hz varyingstrain from 0005 to 05 to determine the limits of the linearviscoelastic region From these strain sweeps a strain of 003was used for subsequent frequency sweeps as it fell within thelinear region for all samples tested The elastic modulus 1198661015840and viscous modulus 11986610158401015840 were then measured at a constantstrain of 003 over a frequency range of 005 to 10HzThe ratioof119866101584010158401198661015840 is called the loss tangent or tan 120575 and is ameasure ofthe ratio of the energy lost to the energy stored in deformation[23]

23 Tear Breakup Time in Rabbits Theexperimental protocoland animal care complied with the NIH Guide for theCare and Use of Laboratory Animals was approved bythe Institutional Animal Care and Use Committee for theUniversity of Utah Tear breakup time (TBUT) was assessedby administering fluorescein dye to the eye and visualizingthe tear film using the cobalt blue function of a slit lampmicroscope TBUT is the time that elapses between the lastblink and the first appearance of breakup in the fluoresceinand is an indicator of tear film stability [24]

International Journal of Biomaterials 3

Table 1 Scores of ocular health before and after 2 weeks of treatment (twice daily) of the xCMHA-S gel in a clinical study on dogs

Case Breed Age(yrs)Gender

STT before(mmmin)

STT after(mmmin)

HyperaemiaBefore

HyperaemiaAfter

IrritationBefore

IrritationAfter

DischargeBefore

DischargeAfter

1 CKCS 8Fn 24 33 22 11 22 00 11 002 Bulldog 6Fn 109 62 22 00 11 00 22 003 Crossbred 10Fn 84 96 21 10 20 00 21 004 St Poodle 11Fn 74 64 22 00 21 00 11 005 CKCS 6Fn 22 33 11 00 11 00 00 006 CKCS 8Fn 33 34 22 00 22 00 11 007 Shih Tzu 12Fn 21 31 22 00 22 00 22 118 Shih Tzu 8Fe 01 22 22 11 22 00 11 009 Bassett 7Mn 49 28 22 11 22 00 00 0010 WHWT 12Fn 21 43 22 11 22 11 11 0111 Lhasa Apso 11Fe 37 26 11 00 11 00 00 0012 ECS 10Mn 32 32 22 00 11 00 11 0013 Eng Setter 7Mn 87 77 11 00 11 00 00 0014 Min Schn 8Fn 98 87 11 00 22 00 00 0015 ECS 9Fe 35 25 11 00 11 00 00 0016 ECS 8Fn 37 26 22 01 22 00 11 0017 WHWT 13Fn 01 22 22 00 22 00 22 1118 WHWT 5Fe 00 12 11 00 11 00 11 0019 Rottweiler 8Me 38 49 22 11 22 11 00 0020 Shih Tzu 7Fn 47 58 21 11 11 00 00 0021 Weimaraner 8Mn 99 812 22 11 22 00 00 0022 ECS 10Fn 45 45 22 00 22 11 00 0023 ECS 12Fn 55 36 11 00 11 00 00 0024 Shih Tzu 7Me 56 66 22 00 11 11 11 0025 Shih Tzu 9Fn 44 54 22 00 22 11 00 00Meanplusmn SD

412 plusmn 288476 plusmn 286





016plusmn037012 plusmn 033



Pvalue 100067 43119864 minus 12

66119864 minus 11

69119864 minus 1214119864 minus 10

50119864 minus 0511119864 minus 02

All scores given are for right eyeleft eye and indicate normal (0) mildly impaired (1) or severely impaired (2) P values indicate comparison between scoresbefore and after treatment In breed CKCS Cavalier King Charles Spaniel St Standard WHWTWest HighlandWhite Terrier ECS English Cocker SpanielEng English Min Schn Miniature Schnauzer In Gender Fn neutered female Fe unaltered female Mn neutered male Me unaltered male STT Schirmertear test

A total of six New Zealand White Rabbits were utilizedin this study Two animals were used as negative controlsand 5 120583L of fluorescein sodium benoxinate hydrochlorideophthalmic solution USP 02504 (Bausch amp Lomb) wasadministered with a micropipette to the corneal surfaceAfter administration the lid was closed manually twice todistribute the tear film and agentThe eyewas then gently heldopen and the TBUT was measured and recorded Both eyesof each animal were used for the negative controlsTheTBUTwas scored by two individuals agreeing and confirming thetime In all cases both individuals who were scoring weremasked to which active compound was being installed

The following day three animals each received eitherxCMHA-S gel or HA2 drops (used as a positive control)For the xCMHA-S and HA2 500120583L of the material wasmixed with 2 drops (100 120583L total) of the fluorescein solution

to create a homogeneous mixture Although addition ofthe fluorescein solution may have reduced the apparentviscosity of each material the amount of fluorescein usedwas necessary for visualization with the slit lamp 50120583L ofthe mixture was then applied to the corneal surface using amicropipette The lid was blinked manually twice as with thenegative control and then gently held open to record TBUT

24 Clinical Evaluation in Dogs with KCS 25 dogs withKCS were included in the study as detailed in Table 1The canine patients were referred to the Queenrsquos Veteri-nary School Hospital Department of Veterinary MedicineUniversity of Cambridge where a diagnosis of KCS wasconfirmed All animals were examined by direct and indirectophthalmoscopy and slit lamp biomicroscopy Ocular surfacehealth was determined as characterized by changes in ocular


Table 2 Properties of the three materials tested in this study The concentration of CMHA-S or HA was determined using a carbazole assay(119899 = 6) Viscosity and shear moduli are averages of 8 samples

Property xCMHA-S HA2 HA4CMHA-S or HA conc (mgmL) 377 plusmn 009 250 plusmn 003 435 plusmn 006Viscosity at 025 sminus1 (Pasdots) 282 plusmn 050 009 plusmn 003 014 plusmn 003Viscosity at 25 sminus1 (Pasdots) 066 plusmn 006 017 plusmn 001 021 plusmn 0011198661015840 at 25Hz (Pa) 414 plusmn 123 446 plusmn 147 325 plusmn 11911986610158401015840 at 25Hz (Pa) 875 plusmn 121 437 plusmn 070 639 plusmn 180

tan 120575 at 025Hz 07 24 16tan 120575 at 5Hz 28 12 25

01

1

10

01 1 10 100

xCMHA-SHA2HA4

Visc

osity

(Pamiddot

s)

Shear rate (1s)

Figure 1 Steady shear viscosity of the crosslinked CMHA-S gel(xCMHA-S) and noncrosslinked HA solutions (HA2 and HA4) asa function of shear rate

hyperaemia ocular irritation and ocular discharge each ofthese clinical features scored as normal (0) mildly impaired(1) or severely impaired (2) Tear production was assessedusing the Schirmer tear test (STT) in which normal canineeyes yield strip wetting of between 15 and 20mm in oneminute but eyes with strip wetting of less than 10mm in oneminute are classified as being mildly affected by KCS andthose with strip wetting of less than 5mmmin are consideredseverely affected Dogs were reexamined after two weeks oftwice daily treatmentwith the xCMHA-S gel and assessmentsof ocular surface health were repeated Data were comparedwith those from a previous study of 25 dogs treated with HA2drops [25]

25 Statistical Analysis Reported 119875 values were determinedusing unpaired two-tailed Studentrsquos 119905-tests betweenmaterialsfor both rheological studies and the TBUT study Mann-Whitney 119880 tests were used to compare the categorical scoredata on improvements in ocular surface health comparingresults before and after treatment as well as comparingtreatment with the xCMHA-S gel and HA2 drops Averagesare reported as mean plusmn standard deviation

3 Results

31 Rheological Assessment To determine the effect of cova-lently crosslinking modified HA on material properties theviscosity and shear moduli of the xCMHA-S gel and a non-crosslinked solution of HA at a similar concentration (HA4)were determined These were compared to a commercial eyeproduct (HA2) that is also a noncrosslinked solution of HAbut at a lower concentration (see Table 2)

Steady shear viscosities of the threematerials as a functionof shear rate are shown in Figure 1 There is a distinct differ-ence in the behavior of the xCMHA-S gel compared to HA2and HA4 and the noncrosslinked HA solutionsThe viscosityof the xCMHA-S gel decreases with increasing shear rateindicating a shear thinning behavior The noncrosslinkedHA solutions on the other hand do not shear thin butrather displayNewtonian behavior Additionally the viscosityof the xCMHA-S gel at shear rates of 025 and 25 sminus1 issignificantly greater than the viscosities of the noncrosslinkedHA solutions (see Table 2)

The elastic 1198661015840 and viscous 11986610158401015840 moduli as a function offrequency are shown in Figures 2(a) and 2(b) respectivelyAt low frequencies below about 1Hz the xCMHA-S gelhas a significantly greater elastic modulus than the othertwo materials However at higher frequencies their elasticmoduli are similar This is also seen in Table 2 whencomparing the elastic moduli for each material at 25HzThe viscous modulus on the other hand is always greaterfor the xCMHA-S gel than the noncrosslinked HA solutionsillustrated in Table 2 for 11986610158401015840 at 25Hz Additionally for thexCMHA-S gel at low frequencies1198661015840 and11986610158401015840 are very similarwhereas at frequencies above approximately 2Hz 11986610158401015840 gt 1198661015840For the noncrosslinked HA solutions 11986610158401015840 gt 1198661015840 over much ofthe frequency range except between about 1 and 5Hz forHA2and about 03 and 2Hz for HA4 when1198661015840 and11986610158401015840 are roughlyequal Loss tangents for these materials at 05 and 5Hz arealso provided in Table 2

32 Tear Breakup Time in Rabbits TBUT was assessed withfluorescein and slit lamp evaluation The xCMHA-S geldeveloped here was compared to a positive control (HA2)and a negative control (fluorescein alone) The applicationof xCMHA-S gel drops increased TBUT compared to HA2(93plusmn12 sec and 71plusmn7 sec resp) although the difference wasnot statistically significant (119875 = 0056 119899 = 3) Both of these


01

1

10

100

001 01 1 10Frequency (Hz)

xCMHA-SHA2HA4

G998400

(Pa)

(a)

01

1

10

100


xCMHA-SHA2HA4

G998400998400

(Pa)

(b)

Figure 2 (a) Elasticmodulus1198661015840 and (b) viscousmodulus11986610158401015840 of the crosslinkedCMHA-S gel (xCMHA-S) and noncrosslinkedHA solutions(HA2 and HA4) as a function of frequency

treatments resulted in significantly longer TBUTs comparedto negative control (38 plusmn 2 sec 119875 lt 00005 for each) TBUTswere evaluated after only one application of each materialUpon application of all study agents eyes were both whiteand quiet After thorough and repeated observation at 24hours postprocedure no eyes showed any sign of irritationor intolerability to any of the applied study agents

33 Clinical Evaluation in Dogs with KCS The mean STTreadings before and after two weeks of treatment for the25 dogs on the xCMHA-S gel are given in Table 1 togetherwith scores for conjunctival hyperaemia ocular irritationand ocular discharge The mean STT readings were notsignificantly changed from pre- to posttreatment howevercategorical scores of ocular surface health were significantlyimproved posttreatment When compared with values previ-ously reported for HA2 [25] improvements in conjunctivalhyperaemia ocular irritation and ocular discharge weresignificantly better for the xCMHA-S gel with 119875-values of0016 and 0006 for conjunctival hyperaemia in the right andleft eyes respectively 0052 and 0053 for irritation in rightand left eyes and 002 and 008 for ocular discharge again inright and left eyes respectively

4 Discussion

KCS is a disease that requires lifelong treatment to miti-gate potential corneal damage and vision loss which oftenincludes the application of a tear supplement formoisture andlubrication Currently available tear replacementmedicationsoften have to be applied several times daily A longer actingtear supplement that could subsequently be applied less fre-quentlymay be beneficial for both patients and owners of petswith KCS Here we have developed an HA-based crosslinkedgel as an extended KCS treatment We characterized the

viscosity and shear moduli of the gel and compared themto two different solutions of noncrosslinked HA We alsodetermined the effect of applying the xCMHA-S gel to thecornea on tear breakup timeWe then used the xCMHA-S gelin a clinical setting treating dogs diagnosed with KCS

Due to the viscoelastic nature of HA as well as the factthat it is anionic it is important to assess the rheologicproperties of HA-based materials In solutions of HA themolecular weight concentration of HA and the presenceof salts or other charged molecules such as proteins canaffect the viscosity and shear moduli Additionally covalentlycrosslinking the HA to form hydrogels can further impactthese rheologic propertiesThe xCMHA-S gel developed hereismade using amodifiedHA inwhich twomodification havebeen made The first modification attaches carboxyl groupsto some of the hydroxyl groups The second modificationattaches thiol groups to some of the carboxyl groups Thesethiol groups are then utilized for the covalent crosslink-ing forming disulfide bonds We have therefore not onlycrosslinked themodifiedHA but also havemodified the ionicnature of themolecule as well Further it should be noted thatduring the crosslinking process the material is continuouslymixed using a whisk attachment with a planetary mixer Thisresults in a material that is not a single continuous gel butrather appears to be effectively a collection ofmicrogels basedon both its physical appearance and its rheological properties

The xCMHA-S gel was found to have a steady shearviscosity that decreased with increasing shear rate typical ofother hydrogels that display shear thinning (pseudoplastic)properties [26] It is also similar to the viscosity behaviorobserved with methacrylated HA near gels and microgels[27] as well as HA-containing materials in the body includ-ing synovial fluid and the vitreous [28 29]The ability to shearthinmay be particularly important for comfort on the cornealsurface allowing the gel to thin during a blinkThis behaviorwas not seen with either the commercial eye lubricant (HA2)


a solution of HA at about 02 nor with another HAsolution (HA4) at about 04 These solutions displayedtypical Newtonian behavior with a roughly constant viscosityover much of the range of shear rates used here similarin behavior to previous results for HA solutions with lowconcentrations [27 30] Although themolecular weight of theHA used in HA2 was not determined it is likely much higherthan the HA used for HA4 here (900 kDa) and the HA usedin a previous study (16MDa) [27] based on their viscositiesAdditionally given the fact that the xCMHA-S gel and theHA4 had roughly the same concentration (about 04) in thesame buffer (PBS) the change in behavior for the viscositycan be attributed primarily to the crosslinked nature of thexCMHA-S gel Although it might have been interesting tocompare the xCMHA-S gel to a noncrosslinked solution ofthe CMHA-S in order to have the samemolecular weight andionic properties this would have been very difficult to achieveand test given that the disulfide crosslinking occurs in thepresence of oxygen at the pH used hereThus we would havehad to either remove the dissolved oxygen from the solutionand perform the rheological testing under argon or nitrogenor lower the pHwhichwould have changed ionic interactions

In addition to determining the viscosity of the materialswe evaluated the elastic and viscous shear moduli under con-stant strain It is important to evaluate these materials underconditions of small-amplitude oscillation as well since thesteady-state viscosity determinations at high shear rates canlead to distortion of a large molecule such as HA and struc-tural breakdownof a gel [31] For the xCMHA-S gel the elasticcomponent is slightly greater than the viscous component atlow frequencies however at high frequencies the materialbehaves more as a viscous solution This is highlighted bythe change in the loss tangent from 07 to 28 at frequenciesof 025 and 5Hz respectively A very similar rheologicalbehavior was observed for methacrylated HA microgels atthe higher volume fractions used [27] and poly-NIPAAmmicrogels [32] Other crosslinked HA gels have displayedmore elastic behavior with 1198661015840 gt 11986610158401015840 over the same rangeof frequencies [26 29] however these gels typically have ahigher concentration of HA and a higher crosslink densitythan the xCMHA-S gel developed here The HA solutionson the other hand demonstrated a predominantly viscousbehavior throughout much of the frequency range withtan 120575 gt 1 at both high and low frequencies for each solutionA similar behavior has been found for other HA solutions atlow concentration or with lower molecular weight [28 30]

TBUT is a standardmeasure employed to evaluate topicaldry eye supplements and the stability of tear filmon the ocularsurface [33] Both the xCMHA-S gel and HA2 extended theTBUT compared to a negative control indicating that bothare able to stabilize the tear film Further the xCMHA-S gelappeared to have a greater effect on the corneal tear film com-pared to HA2 Although the 119875-value (0056) did not reachthe level of statistical significance this was likely due to thelow number of animals used (119899 = 3) and the results suggesta potential benefit of using a crosslinked gel to treat KCSAdditionally the rabbits used for this study had a normal tearfilm and thus were not a dry eye disease model Neverthelessthe results of this study correspond well with previous studies

that indicate the presence of HA which stabilizes the cornealtear film [34ndash36] and these results indicate that a crosslinkedform may enhance this more than a noncrosslinked formIt is not clear from the present study however whetherthe enhanced effect is due to the differences in viscosity orrheological properties found between the xCMHA-S gel andthe noncrosslinked HA solutions or some other mechanisms

With the promising results observed in the rheologicalassessment andTBUT study for the xCMHA-S gel wewantedto determine whether this formulation could be used onlytwice daily in a clinical setting and result in improvementsin the symptoms associated with KCS in dogs The xCMHA-S gel significantly improved the characteristic signs of caninekeratoconjunctivitis sicca in two weeks when given on thisdosing schedule a reduced dosing schedule compared tomany other tear supplements [4 36] Although the STT valuedid not change from pre- to posttreatment we did not expectit to since the material acting as a tear supplement shouldnot stimulate an increase in tear production itself but ratherimprove the symptoms associated with KCS Comparisonwith a previous study [25] shows that the improvements seenwith the crosslinked modified HA are significantly betterthan with a standard noncrosslinked solution of HA (HA2)Clearly there may be potential problems with comparingtwo different groups of dogs however the pretreatment STTvalues and scores for conjunctival hyperaemia ocular irri-tation and ocular discharge were not significantly different(119875 always gt005) between the groups of dogs suggesting thatthe two groups were similar enough for valid comparisonsto be made We also recognize that the current clinical studywas unmasked and therefore could have led to some bias yetwe consider these very promising results A blinded study iscurrently underway directly comparing the two tear replace-ment formulations in KCS-affected dogs

We have produced a crosslinked HA-based hydrogelformulation intended to be used in relieving the symptoms ofKCS This crosslinked version of HA has significantly differ-ent viscosity and rheological properties than correspondingnoncrosslinked solutions of HA Additionally this gel is ableto stabilize the tear film potentially better than a simplesolution of HA and was able to lead to improvements in KCSsymptoms in dogs in a preliminary clinical setting within twoweeks despite being used only twice daily Such a formulationmay lead to better patient eye health and owner complianceThis formulation not only has relevance for the pet dog pop-ulation but also uses these animals as a naturally occurringspontaneous model for human dry eye a significant problemwhere the drive for better tear replacements continues apace

Conflict of Interest

D L Williams declares no conflict of interests B K Mann isemployed by and owns stock in SentrX Animal Care wherethe xCMHA-S formulation used in this study was developed

Acknowledgments

The authors would like to thank Dr Barbara WirostkoBonnie Anderson and R Michael Burr at the Moran Eye


Center University ofUtah for their assistancewith the TBUTstudy

References

[1] J A Smith ldquoThe epidemiology of dry eye disease report ofthe epidemiology subcommittee of the international Dry EyeWorkShop (2007)rdquoOcular Surface vol 5 no 2 pp 93ndash107 2007

[2] H D Perry ldquoDry eye disease pathophysiology classificationand diagnosisrdquo American Journal of Managed Care vol 14 no3 pp S79ndashS87 2008

[3] M E Stern J Gao K F Siemasko R W Beuerman and SC Pflugfelder ldquoThe role of the lacrimal functional unit in thepathophysiology of dry eyerdquo Experimental Eye Research vol 78no 3 pp 409ndash416 2004

[4] D L Williams ldquoImmunopathogenesis of keratoconjunctivitissicca in the dogrdquo Veterinary Clinics of North America vol 38no 2 pp 251ndash268 2008

[5] R F Sanchez G Innocent J Mould and F M Billson ldquoCaninekeratoconjunctivitis sicca disease trends in a review of 229casesrdquo Journal of Small Animal Practice vol 48 no 4 pp 211ndash217 2007

[6] C Hartley D L Williams and V J Adams ldquoEffect of agegender weight and time of day on tear production in normaldogsrdquo Veterinary Ophthalmology vol 9 no 1 pp 53ndash57 2006

[7] M A Lemp ldquoManagement of dry eye diseaserdquo AmericanJournal of Managed Care vol 14 no 3 pp S88ndashS101 2008

[8] M Ali and M E Byrne ldquoControlled release of high molecularweight hyaluronic acid from molecularly imprinted hydrogelcontact lensesrdquo Pharmaceutical Research vol 26 no 3 pp 714ndash726 2009

[9] B P Toole ldquoHyaluronan in morphogenesisrdquo Journal of InternalMedicine vol 242 no 1 pp 35ndash40 1997

[10] J R E Fraser T C Laurent and U B G Laurent ldquoHyaluronanits nature distribution functions and turnoverrdquo Journal ofInternal Medicine vol 242 no 1 pp 27ndash33 1997

[11] E A Balazs ldquoSodium hyaluronate and viscosurgeryrdquo inHealonA Guide to Its Use in Ophthalmic Surgery D Miller and RStegmann Eds pp 5ndash28 John Wiley amp Sons New York NYUSA 1983

[12] K Tsubota and M Yamada ldquoTear evaporation from the ocularsurfacerdquo Investigative Ophthalmology andVisual Science vol 33no 10 pp 2942ndash2950 1992

[13] T Hamano K Horimoto M Lee and S Komemushi ldquoSodiumhyaluronate eyedrops enhance tear film stabilityrdquo JapaneseJournal of Ophthalmology vol 40 no 1 pp 62ndash65 1996

[14] F Guillaumie P Furrer O Felt-Baeyens et al ldquoComparativestudies of various hyaluronic acids produced by microbialfermentation for potential topical ophthalmic applicationsrdquoJournal of Biomedical Materials Research A vol 92 no 4 pp1421ndash1430 2010

[15] S J Falcone D M Palmeri and R A Berg ldquoRheological andcohesive properties of hyaluronic acidrdquo Journal of BiomedicalMaterials Research A vol 76 no 4 pp 721ndash728 2006

[16] D A Gibbs E W Merrill K A Smith and E A BalazsldquoRheology of hyaluronic acidrdquo Biopolymers vol 6 no 6 pp777ndash791 1968

[17] Y Kobayashi A Okamoto and K Nishinari ldquoViscoelasticity ofhyaluronic acid with different molecular weightsrdquo Biorheologyvol 31 no 3 pp 235ndash244 1994

[18] T Higashide and K Sugiyama ldquoUse of viscoelastic substance inophthalmic surgerymdashfocus on sodium hyaluronterdquo Journal ofClinical Ophthalmology vol 2 pp 21ndash30 2008

[19] M J Rah ldquoA review of hyaluronan and its ophthalmic applica-tionsrdquo Optometry vol 82 no 1 pp 38ndash43 2011

[20] G Yang G D Prestwich and B K Mann ldquoThiolatedcarboxymethyl hyaluronic acid-based biomaterials enhancewound healing in rats dogs and horsesrdquo ISRN VeterinaryScience vol 2011 Article ID 851593 7 pages 2011

[21] G Yang L EspandarNMamalis andGD Prestwich ldquoA cross-linked hyaluronan gel accelerates healing of corneal epithelialabrasion and alkali burn injuries in rabbitsrdquoVeterinaryOphthal-mology vol 13 no 3 pp 144ndash150 2010

[22] N S Fedarko ldquoPurification of proteoglycans from mineralizedtissuesrdquoMethods in Molecular Biology vol 171 pp 19ndash25 2001

[23] J D Ferry Viscoelastic Properties of Polymers John Wiley ampSons New York NY USA 3rd edition 1980

[24] A Shafiee C Bucolo E Budzynski K W Ward and F JLopez ldquoIn vivo ocular efficacy profile of mapracorat a novelselective glucocorticoid receptor agonist in rabbit models ofocular diseaserdquo Investigative Ophthalmology and Visual Sciencevol 52 no 3 pp 1422ndash1430 2011

[25] D L Williams S Middleton H Fattahian and R MoridpourldquoComparison of hyaluronic acid-containing topical eye dropswith carbomer-based topical ocular gel as a tear replacement incanine keratoconjunctivitis sicca a prospective study in twenty-five dogsrdquo Veterinary Research Forum vol 3 pp 229ndash232 2012

[26] R Barbucci S Lamponi A Borzacchiello et al ldquoHyaluronicacid hydrogel in the treatment of osteoarthritisrdquo Biomaterialsvol 23 no 23 pp 4503ndash4513 2002

[27] J E Prata T A Barth S A Bencherif and N R WashburnldquoComplex fluids based on methacrylated hyaluronic acidrdquoBiomacromolecules vol 11 no 3 pp 769ndash775 2010

[28] M Mensitieri L Ambrosio S Iannace L Nicolais and A Per-bellini ldquoViscoelastic evaluation of different knee osteoarthritistherapiesrdquo Journal of Materials Science vol 6 no 3 pp 130ndash1371995

[29] M Mensitieri L Ambrosia L Nicolais L Balzano and DLepore ldquoThe rheological behaviour of animal vitreus andits comparison with vitreal substitutesrdquo Journal of MaterialsScience vol 5 no 9-10 pp 743ndash747 1994

[30] A Borzacchiello and L Ambrosio ldquoNetwork formation oflow molecular weight hyaluronic acid derivativesrdquo Journal ofBiomaterials Science Polymer Edition vol 12 no 3 pp 307ndash3162001

[31] R Lapasin S Pricl and P Tracanelli ldquoDifferent behaviors ofconcentrated polysaccharide systems in large-amplitude oscil-lating shear fieldsrdquo Rheologica Acta vol 31 no 4 pp 374ndash3801992

[32] D M Ole Kiminta P F Luckham and S Lenon ldquoThe rheologyof deformable and thermoresponsive microgel particlesrdquo Poly-mer vol 36 no 25 pp 4827ndash4831 1995

[33] G Savini P Prabhawasat T Kojima M Grueterich E Espanaand E Goto ldquoThe challenge of dry eye diagnosisrdquo Journal ofClinical Ophthalmology vol 2 pp 31ndash55 2008

[34] S Nakamura S Okada Y Umeda and F Saito ldquoDevelopmentof a rabbit model of tear film instability and evaluation ofviscosity of artificial tear preparationsrdquo Cornea vol 23 no 4pp 390ndash397 2004


[35] HMochizukiM Yamada S Hato and TNishida ldquoFluoropho-tometric measurement of the precorneal residence time of topi-cally applied hyaluronic acidrdquo British Journal of Ophthalmologyvol 92 no 1 pp 108ndash111 2008

[36] L C McCann A Tomlinson E I Pearce and V Papa ldquoEffec-tiveness of artificial tears in the management of evaporative dryeyerdquo Cornea vol 31 no 1 pp 1ndash5 2012

Submit your manuscripts athttpwwwhindawicom

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supplement on the other hand is often administered fourtimes daily or more

Tear supplements often contain a compound to increasethe viscosity of the solution such as polyvinyl alcoholhydroxypropylmethylcellulose carboxymethylcellulose pol-yethylene glycol or hyaluronic acid (HA) [4 7 8] HA isparticularly attractive as it is a naturally occurring polysac-charide found throughout the body during all stages ofdevelopment [9 10] In the eye HA is in the aqueous humorand vitreous and also coats the corneal endothelium [11]In tear supplements the viscoelasticity of HA leads to areduction of tear removal and an increase in tear stabilitythereby reducing some of the symptoms of dry eye [12 13]

Due to this viscoelasticity one means of characterizingHA-based products is to determine the rheologic propertiesof the eye drop or gelThese properties are important to assesssince the molecular weight and concentration of the HA willaffect the viscosity as well as elastic and viscous shear moduliof the solution [14 15] Additionally the concentration ofions such as salts in the solutionmay influence the rheologicproperties due to the polyanionic nature of HA [16 17]Several HA-based products with varying rheologic propertieshave been developed for ophthalmic surgery [18 19] andthese properties may influence comfort and efficacy in a dryeye formulation [14]

Although HA-based tear supplements have been used forover 20 years a formulation that extends the contact time ofthe HA with the ocular surface may allow for less frequentapplication reducing the overall cost and burden on thepatient and in the case of dogs the owner One method ofextending the contact timemay be to covalently crosslink theHA rather than the physical or ionic crosslinking that occursin solutions of high molecular weight HA A modified HAthiolated carboxymethyl HA (CMHA-S) has previously beenused to create crosslinked hydrogels for treating skin andcorneal wounds in multiple species [20 21] Here we havedeveloped a new hydrogel formulation as a tear supplementfor treating KCS in dogs The hydrogel was characterizedusing rheology to compare to noncrosslinked solutions ofHA The crosslinked CMHA-S hydrogel was then used ina clinical setting to treat dogs previously diagnosed withKCS monitoring response to therapy by evaluating tearproduction (Schirmer tear test) conjunctival hyperaemiaocular discharge and ocular irritation as determined byblink frequency and palpebral aperture narrowing With thisnew crosslinked hydrogel formulation a reduction in bothrequired application frequency and in KCS symptoms isanticipated and should improve both patient corneal healthand owner compliance

2 Materials and Methods

21 Crosslinked CMHA-S Hydrogel CMHA-S was syn-thesized as previously described [21] Thiol modification(3 times 10minus4mmolmg) was assessed using 551015840-dithio-bis(2-nitrobenzoic acid) (Ellmanrsquos reagent Sigma-Aldrich) MW(340 kDa) was assessed using gel permeation chromatog-raphy and dynamic light scattering Purified CMHA-S was

diluted to a final concentration of 4mgmL in phosphate-buffered saline (PBS pH 74 Fisher Scientific) The CMHA-Ssolution was then sterilized through a 02120583m filter intoa sterile mixing bowl The solution was crosslinked usinga dilute solution of sodium hypochlorite (Sigma-Aldrich)while mixing and the resultant gel was mixed overnightThe crosslinkedCMHA-S gel (xCMHA-S) was then packagedaseptically into sterile 15mL eye drop bottles A solution ofnoncrosslinked HA (900 kDa Novozymes) at approximately4mgmL in PBS (hereinafter referred to as HA4) was madefor comparison in the rheological studies below

To verify the final concentration of CMHA-S in the pack-aged xCMHA-S gel and HA in the noncrosslinked solution(HA4) a carbazole assay was used which detects uronic acid[22]TheHA concentration in a commercial eye product thatis a noncrosslinked solution of HA (Clinadry Eye LubricantIDPHAR Belgian Pharmaceuticals) (hereinafter referred toas HA2) was also assessed using this assay Briefly 250 120583Lof sample or standard was combined with 15mL ice-cold0025M sodium tetraborate decahydrate (Sigma-Aldrich) inconcentrated sulfuric acid (Acros Organics) and then heatedin boiling water for 10min followed by rapid cooling in an icebath 50120583L of 0125 carbazole (Sigma-Aldrich) in analyticalgrade methanol (Acros Organics) was added to each sampleand standard The mixtures were heated in boiling water for15min and then cooled rapidly to room temperature Theabsorbance at 530 nm was measured on a spectrophotometer(Genesys 10S Thermo Scientific) Six samples were used foreach formulation standards used here were solutions of HAin PBS

22 Rheological Assessment Rheological testing was per-formed on an AR1000 rheometer (TA Instruments) using a25mm diameter parallel plate format Samples (5-6mL) wereplaced in a 35mm plastic Petri dish and the rheometer headlowered to a gap distance of 5mm To determine viscosityshear rate was varied from 01 to 100 sminus1 A strain sweep wasthen performed at an oscillation frequency of 1Hz varyingstrain from 0005 to 05 to determine the limits of the linearviscoelastic region From these strain sweeps a strain of 003was used for subsequent frequency sweeps as it fell within thelinear region for all samples tested The elastic modulus 1198661015840and viscous modulus 11986610158401015840 were then measured at a constantstrain of 003 over a frequency range of 005 to 10HzThe ratioof119866101584010158401198661015840 is called the loss tangent or tan 120575 and is ameasure ofthe ratio of the energy lost to the energy stored in deformation[23]

23 Tear Breakup Time in Rabbits Theexperimental protocoland animal care complied with the NIH Guide for theCare and Use of Laboratory Animals was approved bythe Institutional Animal Care and Use Committee for theUniversity of Utah Tear breakup time (TBUT) was assessedby administering fluorescein dye to the eye and visualizingthe tear film using the cobalt blue function of a slit lampmicroscope TBUT is the time that elapses between the lastblink and the first appearance of breakup in the fluoresceinand is an indicator of tear film stability [24]




STT before(mmmin)

STT after(mmmin)

HyperaemiaBefore

HyperaemiaAfter

IrritationBefore

IrritationAfter

DischargeBefore

DischargeAfter










Pvalue 100067 43119864 minus 12

66119864 minus 11

69119864 minus 1214119864 minus 10

50119864 minus 0511119864 minus 02









tan 120575 at 025Hz 07 24 16tan 120575 at 5Hz 28 12 25

01

1

10

01 1 10 100

xCMHA-SHA2HA4

Visc

osity

(Pamiddot

s)

Shear rate (1s)




3 Results






01

1

10

100


xCMHA-SHA2HA4

G998400

(Pa)

(a)

01

1

10

100


xCMHA-SHA2HA4

G998400998400

(Pa)

(b)




4 Discussion














Acknowledgments




References













































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials






STT before(mmmin)

STT after(mmmin)

HyperaemiaBefore

HyperaemiaAfter

IrritationBefore

IrritationAfter

DischargeBefore

DischargeAfter










Pvalue 100067 43119864 minus 12

66119864 minus 11

69119864 minus 1214119864 minus 10

50119864 minus 0511119864 minus 02









tan 120575 at 025Hz 07 24 16tan 120575 at 5Hz 28 12 25

01

1

10

01 1 10 100

xCMHA-SHA2HA4

Visc

osity

(Pamiddot

s)

Shear rate (1s)




3 Results






01

1

10

100


xCMHA-SHA2HA4

G998400

(Pa)

(a)

01

1

10

100


xCMHA-SHA2HA4

G998400998400

(Pa)

(b)




4 Discussion














Acknowledgments




References













































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials






tan 120575 at 025Hz 07 24 16tan 120575 at 5Hz 28 12 25

01

1

10

01 1 10 100

xCMHA-SHA2HA4

Visc

osity

(Pamiddot

s)

Shear rate (1s)




3 Results






01

1

10

100


xCMHA-SHA2HA4

G998400

(Pa)

(a)

01

1

10

100


xCMHA-SHA2HA4

G998400998400

(Pa)

(b)




4 Discussion














Acknowledgments




References













































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials




01

1

10

100


xCMHA-SHA2HA4

G998400

(Pa)

(a)

01

1

10

100


xCMHA-SHA2HA4

G998400998400

(Pa)

(b)




4 Discussion














Acknowledgments




References













































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials












Acknowledgments




References













































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials





References













































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials













CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials



Research Article A Crosslinked HA-Based Hydrogel...

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