Biomimetic hydrogels gate transport of calcium ionsacross cell culture inserts

Christian N. Kotanen & A. Nolan Wilson &

Ann M. Wilson & Kazuhiko Ishihara &

Anthony Guiseppi-Elie

Published online: 17 March 2012# Springer Science+Business Media, LLC 2012

Abstract Control of the in vitro spatiotemporal availability ofcalcium ions is one means by which the microenvironments ofhematopoietic stem cells grown in culture may be reproduced.The effects of cross-linking density on the diffusivity of calci-um ions through cell culture compatible poly(2-hydroxyethylmethacrylate) [poly(HEMA)]-based bioactive hydrogels pos-sessing 1.0 mol% 2-methacryloyloxyethyl phosphorylcholine(MPC), 5 mol% N,N-(dimethylamino)ethylmethacrylate(DMAEMA) and ca. 17 mol% n-butyl acrylate (n-BA) have

been investigated to determine if varying cross-link density is aviable approach to controlling transport of calcium acrosshydrogel membranes. Cross-linking density was varied bychanging the composition of cross-linker, tetraethyleneglycoldiacrylate (TEGDA). The hydrogel membranes were formedby sandwich casting onto the external surface of track-etchedpolycarbonate membranes (T010 μm, φ00.4 μm pores) ofcell culture inserts, polymerized in place by UV light irradia-tion and immersed in buffered (0.025 HEPES, pH 7.4) 0.10 Mcalcium chloride solution. The transport of calcium ions acrossthe hydrogel membrane was monitored using a calcium ionselective electrode set within the insert. Degree of hydration(21.6±1.0%) and void fraction were found to be constantacross all cross-linking densities. Diffusion coefficients, deter-mined using time-lag analysis, were shown to be stronglydependent on and to exponentially decrease with increasingcross-linking density. Compared to that found in buffer (2.0–2.5×10−6 cm2/s), diffusion coefficients ranged from 1.40×10−6 cm2/s to 1.80×10−7 cm2/s and tortuosity values rangedfrom 1.7 to 10.0 for the 1 and 12 mol% TEGDA cross-linkedhydrogels respectively. Changes in tortuosity arising fromvariations in cross-link density were found to be the primarymodality for controlling diffusivity through novel n-BAcontaining poly(HEMA)-based bioactive hydrogels.

Keywords Poly(HEMA) . Biomimetic . Hydrogels .

Co-networks .Modulus . Cross-linking density

1 Introduction

Polymer hydrogels (Ottenbrite et al. 2010) are three-dimensional (3-D) networks synthesized from highly hydro-philic monomers rendered insoluble by virtual, electrostaticor covalent cross-linking (Mack et al. 1988). Hydrogels

Christian N. Kotanen and A. Nolan Wilson contributed equallytowards this work.

C. N. Kotanen :A. N. Wilson :A. Guiseppi-Elie (*)Center for Bioelectronics, Biosensors and Biochips (C3B),Clemson University,100 Technology Drive,Anderson, SC 29625, USAe-mail: guiseppi@clemson.edu

C. N. Kotanen :A. Guiseppi-ElieBioengineering, Clemson University,Clemson, SC 29634, USA

A. N. Wilson :A. Guiseppi-ElieChemical and Biomolecular Engineering, Clemson University,Clemson, SC 29634, USA

A. M. WilsonDepartment of Chemistry, University of the West Indies,St. Augustine, Republic of Trinidad and Tobago

K. IshiharaDepartments of Materials Engineering and Bioengineering,School of Engineering, The University of Tokyo,7-3-1, Hongo, Bunkyo-ku,Tokyo 113-8656, Japan

A. Guiseppi-ElieElectrical and Computer Engineering, Clemson University,Clemson, SC 29634, USA

Biomed Microdevices (2012) 14:549–558DOI 10.1007/s10544-012-9632-0

imbibe large amounts of water that may be free or bound(Wang et al. 2008). When immersed and equilibrated in anaqueous medium, cross-linked hydrogels assume their final3-D hydrated network structure which brings into balancetwo classes of forces. The first is the swelling force, whicharises from the solvation of the repeat units of the macro-molecular chains and tends to produce an expansion of themacromolecular network. The second is the counter balanc-ing retractive force which results from the cross-linkedstructure (Metters et al. 2000). The result is an elasticnetwork with water effectively filling the interstices or voidsof the network. The hydrogel can accordingly change itssize and shape by expelling or imbibing free water in re-sponse to environmental stimuli, and this is one of itsintrinsic characteristics (Gawel et al. 2010). Moreover, inso doing, hydrogels may also imbibe other monomeric,reactive, and potentially polymerizable species into its in-terstices which occupy its void volume and can interact withchain segments or pendant moieties of the host hydrogel(Guiseppi-Elie 2010). Their swelling capacity results in highpermeabilities for low molecular weight bioactive molecules(Lin and Metters 2006; Trigo et al. 1993), metabolites (Li etal. 2004), and redox mediators (Boztas and Guiseppi-Elie2009). These characteristics have allowed hydrogels to beused in biomedical applications that include biosensors andbiochips (Yu et al. 2008), drug delivery systems (Kopeček2010), scaffolds in regenerative medicine (Slaughter et al.2009), as base materials for soft-contact lenses (Russo et al.2007), and as tourniquets (Wilson et al. 2010). Hydrogelshave emerged as one of the most widely researched, patent-ed, and successfully commercialized biomedical polymers(Alfrey et al. 1966). When implanted, the soft elastomericnature of hydrogels serves to minimize mechanical andfrictional irritation to the hosting tissue bed and their lowinterfacial tension contributes to a reduction in proteinadsorption/denaturation and hence biofouling.

Hydrogel constructs capable of interfacing with nativebiological tissue through physical and chemical semblanceare being molecularly engineered to alter cell migration,growth, proliferation, differentiation, morphology, and proteinexpression. Hydrogels are also being molecularly engineeredto elicit these same effects through the programmed release ofbiomolecules. In both cases, transport properties allow fortime dependent release and exposure to biological entities.One means by which biomolecule transport can be controlledis through the degree of hydration within hydrogels. It hasbeen previously observed that crosslink density of hydrogels,through its influence on the degree of hydration, systematical-ly controls diffusivity (Boztas and Guiseppi-Elie 2009). Co-valent cross linking within acrylate based hydrogels is easilyperformed using bi-functional monomers like tetraethylene-glycol diacrylate. However, for in vivo purposes, the design ofthe hydrogel construct must be specially tailored to suit the

biomolecule of interest while still maintaining desired bio-compatibility. Novel hydrogels, or biosmart hydrogels, consistof pendant groups or moieties with purpose to impart a bal-ance of hydrophilicity and hydrophobicity, control of glasstransition temperature, pH, temperature and ionic strengthresponsiveness, as well as confer biocompatibility.

Cells are in a constant state of regulatory feedback inresponse to numerous endogenous and exogenous stimuli.Calcium functions as a ubiquitous stimulatory messenger incells, regulating molecular processes that include fertiliza-tion, embryogenesis, muscle contraction, and synaptic ves-icle release to gene transcription and metabolism (Berridgeet al. 2003). As an example, osteoblasts play a crucial role indevelopment and fate of hematopoietic stem cells (HSCs).The expression of Angiopoietins by osteoblasts promotesTie-2 receptors on HSCs enabling their adhesion to niches,maintenance, and self-renewal (Arai et al. 2004; Zhang et al.2003). A change in the calcium ion environment of cellscultured in vitro has been shown to control osteoblast HSCniche-related protein expression (Nakamura et al. 2010).Osteoblasts mineralize, show significant morphologicalchanges, and express Angiopoietins 1 and 2 (Ang1, Ang2)as functions of culture time and calcium concentration in therange of 6–50 mM (Nakamura et al. 2010). Control of thespatiotemporal distribution of calcium ions within cell cul-ture media through the use of transport regulating hydrogelmembranes is one means by which HSC niche-like micro-environments may be reproduced in vitro. Degradable ce-ramic biomaterials are excellent candidates for regulatingcalcium ion release over time (Capuccini et al. 2008; Anadaet al. 2008; Matsumoto et al. 2007). However, systemsbased on degradable scaffolds will not be reversible norcan they adapt and respond to changes in the cell culturemedia. Hydrogel scaffolds are capable of reversible swellingand deswelling behavior, which can be controlled by tem-perature, pH, light, and redox state (Hoffman 2002; ShipwayandWillner 2001). There is a need for programmed regulationof calcium environments leading to idealized HSCmicroenvironments for engineering bone marrow andinducing effective bone regeneration.

In most hydrogels, the degree of hydration will predom-inantly influence diffusivity of molecules (am Ende et al.1995; Bae et al. 1989; Boztas and Guiseppi-Elie 2009;Compañ et al. 1996; Murphy et al. 1988; Pitt et al. 1992;Varshosaz and Falamarzian 2001). However, the influenceof degree of hydration on diffusivity of physiologicallyrelevant molecules has yet to be fully explored for complexbiosmart hydrogels. The current work evaluates the role ofcrosslink density and the degree of hydration on Ca++ trans-port through a biosmart hydrogel. The use of biomimetichydrogels as scaffolds for controlling transport properties ofbiologically relevant solutes such as calcium is investigatedusing time lag analysis.

550 Biomed Microdevices (2012) 14:549–558

2 Experimental

2.1 Reagents

Anhydrous calcium chloride (CaCl2) and surface modifyingreagent, octadecyltrichlorosilane (OTS, 90%), were purchasedfrom Sigma Aldrich. Hydrogel components HEMA, n-butylacrylate (n-BA, 99%), tetraethyleneglycol diacrylate (TEGDA,technical grade), N-[tris(hydroxymethyl)methyl]-acrylamide(HMMA, 93%), poly(ethyleneglycol)(400)monomethacrylate(PEG(400)MA), N,N-(dimethylamino)ethyl methacrylate(DMAEMA, 98%), poly(N-vinylpyrrolidone) (poly(NVP),MW 1.3×106), and the photoinitiator 2,2-dimethoxy-2-phe-nylacetophenone (DMPA, 99%) were all purchased fromSigma-Aldrich. 2-Methacryloyloxyethyl phosphorylcholine(MPC) was synthesized elsewhere as previously described(Ishihara et al. 1990). Prior to formulation, all of the acrylate-containing reagents were passed over an inhibitor removalcolumn (Sigma-Aldrich) to remove the polymerization inhib-itors hydroquinone and monomethyl ether hydroquinone. Tris(hydroxymethyl)aminomethane (TRIS buffer, ACS reagent,99.8+%,) was pH-adjusted with hydrochloric acid (HCl)(ACS reagent, 37%) to obtain 0.1 M buffer with pH07.2.Common solvents were purchased from Sigma Aldrich andused as received. A 0.025 M 4-(2-hydroxyethyl)-1-piperazi-neethanesulfonic acid sodium salt (HEPES) buffer was madeand pH-adjusted to pH07.35 using 1 M HCl. Phosphatebuffered saline (PBS) (0.01 M, pH7.4). All solutions wereprepared with deionized (MilliQ DI) water.

2.2 Formulation of poly(HEMA)-based hydrogel cocktails

Hydrogel cocktails were formulated by mixing the nine con-stituents HEMA, n-BA, TEGMA, PEG(400)MA, HMMA,MPC, poly(NVP), DMAEMA, and DMPA in mole % asdetailed in Table 1. HEMA (hydrophilic) and n-BA (hydro-phobic) were used in a 10:3 mole ratio and sum to 81-α mol%, where α is the mol% TEGDA. PEG(400)MA and poly(NVP) were expressed in mole % of repeat units. Six differentformulations of cross link densities corresponding to α01, 3,5, 7, 9 and 12 mol% TEGDA were prepared for study. Poly(HEMA) is a base hydrogel for in vivo biocompatibility as it isnon-toxic and well tolerated in the body (Jeyanthi andPanduranga Rao 1990; Montheard et al. 1992). The acrylatebased chemistry is an efficient means of incorporating mono-mers with unique pendant groups to poly(HEMA)-basedhydrogels. Radical chain polymerization with DMPA can bephotoinitiated with ultraviolet light for simple one-step poly-merizations. Moieties in MPC and PEG(400)MA togetherhave shown to impart a high resistance to extracellular matrixprotein adsorption (Abraham et al. 2005). These pendantgroups may have synergistic or antagonistic effects as a resultof their concerted actions at the molecular level and so add

confounding complexity that may impact diffusivity ofcontrolled-release biomolecules. Hydrogel constituents suchas DMAEMA can control swelling content based on local pHchanges (Brahim et al. 2003; Guiseppi-Elie et al. 2002). Self-plasticization of acrylate based hydrogels is possible usingmonomers with hydrophobic side groups such as n-BA (Hengand Hall 1999). Hydrophilicity of gels can be increased bydirectly including highly hydrophilic constituents such asHMMAwhich introduce manymore pendant hydroxyl groupsper repeat unit. Poly(NVP) was added to serve as a viscositymodifier, allowing the control of membrane thickness via spincoating. Finally, TEGDAwas used as the cross linking agentand was used in varying mole% to control the degree ofhydration.

Figure 1 shows a schematic of themolecular structure of theseveral constituents used in the preparation of the hydrogelcocktails. To this mixture was then added a 1:1 (v/v) mixedsolvent of ethylene glycol and water so that the mixed solventcomprised 20 wt.% of the final formulation. Under UV-freeconditions, the final hydrogel cocktail was sonicated (5.0 min),mixed overnight, and sparged with nitrogen gas (ca. 30 s).

2.3 Preparation of hydrophobic microscope slides

Hydrophobic glass microscope slides were prepared in ad-vance for use in the preparation of ion transport cells. Slideswere degreased by sequential immersion in boiling trichlo-roethylene, acetone, and isopropyl alcohol for 3 minuteseach, followed by thorough rinsing in DI-water for 1 min.To create a surface of hydroxyl groups, slides were subse-quently treated and activated with RCA clean (1:1:5NH4OH:H2O2(30%):DI-H2O) by immersion for 10 min fol-lowed by rinsing with DI-water. Slides were then silanizedby immersion in a solution of 0.1 wt.% OTS in anhydrous

Table 1 Hydrogel constituents and composition expressed in mol%and mol% of repeat units where noted, HEMA and n-BA were main-tained at a constant 10:3 mol ratio and TEGDA was varied for thehydrogel compositions under test

Constituent Mole fraction (mol%)

a HEMA 61.54 60.00 58.46 56.92 55.39 53.08a n-BA 18.46 18.00 17.54 17.08 16.62 15.92

TEGDA (α) variablecrosslink density

1.00 3.00 5.00 7.00 9.00 12.00

b PEG(400)MA 5.00 5.00 5.00 5.00 5.00 5.00

MPC 1.00 1.00 1.00 1.00 1.00 1.00

HMMA 5.00 5.00 5.00 5.00 5.00 5.00b Poly(NVP) 2.00 2.00 2.00 2.00 2.00 2.00

DMAEMA 5.00 5.00 5.00 5.00 5.00 5.00

DMPA 1.00 1.00 1.00 1.00 1.00 1.00

a Maintained at a 10:3 ratio of HEMA to n-BAbMole % of repeat units

Biomed Microdevices (2012) 14:549–558 551

toluene for 30 min at room temperature followed by ultra-sonication in anhydrous toluene for 1 min. Curing of thenewly formed siloxane layer was performed in a 0.22 μmfiltered convection oven for 20 min each at the followingtemperatures: 40°C, 110°C, and 40°C. Slides were stored dry.

2.4 Preparation of ion transport cells

The ion transport cell was fashioned from cell culture inserts(Model 3401, Corning Life Sciences), Fig. 2. Polycarbonatemembranes of cell culture inserts featured alpha-particletrack etched pores of 0.40 μm in diameter, 108 pores/cm2,an area of 1.12 cm2 and 10.0 μm in thickness. All prepara-tions were made under clean room conditions. Monomercocktail was pipetted (30 μL) onto the external surface ofpolycarbonate membrane of inserts, covered with the hydro-phobic microscope slide to create a uniformly thick, contig-uous liquid layer, and immediately irradiated with UV light(366 nm, 2.3 W/cm2, 5–7 min) in a UV cross-linker (CX-2000, UVP, Upland, CA, USA) under an inert nitrogenatmosphere to effect polymerization of the hydrogel com-ponent. Cross-linked hydrogels were placed in a bath of

HEPES buffer at 37°C for 12 h to hydrate and detach fromthe microscope slides. Finally, un-reacted monomer wasextracted by submerging hydrogel-coated inserts into freshbuffer that was replaced hourly for a total of four hours priorto calcium ion transport studies. Hydrogel thicknesses weremeasured using a digital micrometer (Mitutoyo 293–344).

2.5 Preparation of hydrogel disks and hydration methodology

Aliquots of 30 μL of the hydrogel cocktail were pipettedinto the corresponding test specimen molds and irradiatedwith UV light (366 nm, 2.3 W/cm2, 5–7 min) in a UV cross-linker (CX-2000, UVP, Upland, CA, USA) under an inertnitrogen atmosphere. The hydrogels were then fully hydrat-ed, wet weights were taken, and photographic dimensionalanalysis (PDA) was performed to determine hydrateddimensions. Hydrated dimensions used the method outlinedby Karageorgiou (Karageorgiou and Kaplan 2005); howev-er, only void volumes accessible by water were considered.The hydrogels were then dehydrated in a Caliper-ZymarkTurboVap 500 (Caliper Life Sciences, Hopkinton, MA,USA), dry weights were repeatedly taken to constant

Confersbiocompatibility

Confershigh hydration

Engineers Tg

Adjusts viscosityand confersbiocompatibility

Cross linker

Fig. 1 Schematic of themolecular structure andpre-polymer constituents ofpoly(HEMA)-based hydrogelpossessing MPC, DMAEMA,n-BA and varying crosslinkdensity based on mole fractionof TEGDA

Fig. 2 Corning 3401 cell culture inserts with hydrogel coated nanoporous polycarbonate membranes. Far left) hydrogel-coated inserts aftercrosslinking; center) insert-gel after detachment from hydrophobic slides; far right) side view of hydrogel-coated insert

552 Biomed Microdevices (2012) 14:549–558

weight, and PDA was performed to determine dehydrateddimensions. Using Eqs. 1 and 2, where HG is hydratedhydrogel, DG is dehydrated hydrogel and M is mass, thevoid fraction and degree of hydration were determined as afunction of cross linked density of the hydrogels.

" ¼ MHG �MDG

ρwater

� �=

MHG

ρHG

� �ð1Þ

Degree of hydration ð%Þ ¼ MHG�MDG

MHG� 100% ð2Þ

2.6 Calcium ion-selective electrode characterization

A glass-body, combination calcium ion-selective electrode(ISE) was acquired from Cole-Parmer. The electrode wasconnected to a pH/voltmeter (Fisher Scientific) and an A-Dconverter (DI-158U, DATAQ Instruments, Inc., Ohio) wasused to directly import voltage readings into MicrosoftExcel. A calcium chloride ladder ranging from 1.0 to100 mM was prepared in HEPES buffer at 37°C. Thecalibration curve for the ISE can be seen in Fig. 3. Theslope of the calibration curve revealed an approximate 25–26 mV change per decade of calcium molarity as predictedby the Nernst Equation. Response time of the ISE wasapproximately 0.5 s. The calcium ISE was stored in aCole-Parmer calcium standard solution of 1000 ppm CaCl2in DI-water at room temperature.

2.7 Measurement of Ca2+ transport through hydrogelmembranes

Figure 4 shows the experimental setup wherein a solution of0.10 M CaCl2 prepared in 0.025 HEPES buffer (pH 7.4) was

used as the calcium ion test solution. The test solutionwas kept in a jacketed beaker (150 mL) to maintain aconstant temperature of 37°C using a temperature recir-culator (Ecoline-RE106/E100, Lauda-Brinkmann, Del-ran, NJ). The calcium ISE was initially allowed toequilibrate in the calcium standard solution at 37°C.After rinsing with DI-water, the tip of the ISE wasplaced inside an insert and clipped to reproducibly holdit in place (ISE-insert). The insert was filled with 1 mLof fresh HEPES buffer to be used as the receivingsolution and placed in a beaker of fresh HEPES at37°C to equilibrate. Miniature stir bars were used insidethe test solution and receiving solution to maintainconstant boundary layer rheological characteristics. Priorto each experiment, voltage measurements were taken inthe receiving solution until a constant, baseline voltagewas obtained pertaining to zero calcium concentration.To begin the experiment, the ISE-insert was crank-lowered using a lab jack into the calcium test solutionand the voltage monitored over time. Calcium diffusionthrough the insert membranes and hydrogels was mon-itored as a function of time. Following each experiment,the electrode was thoroughly rinsed with DI-water andallowed to equilibrate in the calcium standard solutionat 37°C in preparation for the next experiment.

3 Results and discussion

3.1 Time lag analysis and apparent diffusion coefficients

When a hydrogel membrane is suspended in a volumeof well stirred test solution that is much larger thanitself, the effect on the bulk solution concentration bythe uptake of the solute into the hydrogel can beconsidered negligible (Crank 1975). The expressionfor the total amount of diffusing substance, Qt, passingthrough the membrane over time, t, during unsteady

10-3

10-2

10-1

40

50

60

70

80

90

100

110

Concentration Ca2+ (M)

Vo

ltag

e (

mV

)

y = 10.933ln(x) + 126.75R² = 0.9911

Fig. 3 Calibration curve of calcium ions, 1.0–100 mM in 0.025 MHEPES at 37°C

Fig. 4 Experimental setup: Inserts filled with fresh HEPES buffer areclipped onto the ISE and lowered into test solution at 37°C. Solutionsare kept well stirred. Voltage readings are taken from the pH/voltmeterthrough an A-D converter and saved to a PC

Biomed Microdevices (2012) 14:549–558 553

state conditions for planar geometry is given by (Crank1975):

Qt ¼ D C1 � C2ð Þ tlþ 21

p2X11

C1 cos npð Þ � C2

n21� exp � Dn2p2t

l2

� �� �

þ 4C0l

p2X1m¼0

1

2mþ 2ð Þ2 1� exp � D 2mþ 1ð Þ2p2tl2

!( )

ð3ÞIn the implementation of this analysis the following

boundary conditions were assumed: i) the face of themembrane in contact with the test solution, x00, has aconstant concentration of the diffusing solute, C1, thatremains constant, ii) the receiving solution side of themembrane, x0 l, the concentration, C2, is constant andapproximately zero, and iii) the initial concentration ofsolute, C0, throughout the membrane is equal to zero.For these given boundary conditions, Eq. 3 can bereduced as follows:

Qt

lC1¼ Dt

l2� 1

6� 2

p2X11

�1ð Þnn2

exp � Dn2p2tl2

� �; ð4Þ

Which, as t→∞, approaches the following form of aline

Qt ¼ DC1

lt � l2

6D

� �; ð5Þ

The intercept of this line with the time axis, tlag, isgiven by

tlag ¼ l2

6Dð6Þ

This method of analysis has been thoroughly described(Crank 1975; Rutherford and Do 1997). Since ISE voltage isdirectly correlated to concentration of solute, the time lagdata of potentiometrically monitored calcium concentrationacross biomimetic hydrogels was used along with Eq. 6 tocalculate diffusion coefficients. Calcium transport data wasanalyzed using MATLAB. Electrode voltage as a functionof time was plotted. Plotting the first derivative of thevoltage curve and locating the maxima yielded the point ofinflection or greatest rate of change in voltage over time fora given data set. A tangent line was fit to the voltage versustime plot at the point of inflection and the time point of theintercept with the reference baseline was used as the mea-sured breakthrough time. Due to the low modulus, accuratemeasurement of hydrated hydrogel thickness using micro-meters is not feasible. By assuming homogenous isotropicswelling of the hydrogels, the hydrated hydrogel thickness,lhydrated, of each crosslink density tested was determined

from the averaged dry thickness, ldry, and the experimentallydetermined degree of hydration:

lhydrated ¼ ldryDegree of hydration %ð Þ

100þ 1

� �; ð7Þ

The hydrated hydrogel thickness and its correspondingbreakthrough time were used to calculate the apparent dif-fusivity of calcium through hydrated hydrogels. The diffu-sion path length, lD, was determined using Eq. 8 and thetortuosity, τ, was calculated using Eq. 9.

lD ¼ StlagV2

A"C1ð8Þ

t ¼ lDlhydrated

ð9Þ

Where S is the steady state slope of the time lag curve,Fig. 5, V2 is the volume of the receiving solution, and A isthe area of the hydrogel perpendicular to the diffusion path.

Statistical analysis was performed using SPSS Statistics17.0 software (SPSS, Chicago, IL). A level of significanceof 0.05 was used for determining confidence intervals,performing analysis of variance and making pairwise com-parisons of sample means. Normality of data was testedusing normal probability plots. Multiple comparisons pro-cedures were performed on data of varying crosslink densi-ty. For pairwise comparisons of samples with samples ofequal number and equal variance, the Tukey HSD test wasapplied. For pairwise comparison of samples with samplesof unequal number and equal variance, the Tukey-Kramertest was applied.

y = 2.126x - 11.44

0

0.5

1

1.5

2

2.5

-10

0

10

20

30

40

50

60

70

0 10 20 30 40 50 60

dV/d

t

Vol

tage

(mV

)

Time (s)

Voltage

dV/dt

Reference baseline

Fig. 5 The first derivative with respect to time of the voltage isdetermined using MATLAB. The maximum point of inflection of thefirst derivative is used to determine the steady state influx of calciumions through the hydrogel membranes. The line tangent to the voltageversus time curve is determined and its intercept with baseline potentialis used as the time lag or breakthrough time

554 Biomed Microdevices (2012) 14:549–558

Table 2 summarizes the results obtained. Polycarbonatemembrane thickness (10 μm) was subtracted from microm-eter measurements of the insert-supported hydrogel mem-brane composite. The poly(HEMA)-based hydrogelsshowed good attachment to the polycarbonate membranes.No disbondment between hydrogels and inserts was ob-served throughout the study. Insert membranes exhibitingmechanical failure, in the form of visible tears due to straininduced during hydrogel swelling, were not used in furtherexperiments. No significant difference in dry thickness val-ues was observed (p00.162) as a function of crosslinkdensity. An average dry thickness (ldry) of 25.4 μm wasused for estimating hydrated thickness values. Appreciablebut constant hydration, averaging 21.6±1.0%, accompaniedthe swelling of the hydrated hydrogels, Table 2.

The breakthrough time of calcium ions through insertswith no cast-applied hydrogel membrane was averaged at6.1±0.1 s with 95% confidence and this value was smallcompared to other breakthrough times and was subtractedfrom breakthrough times measured for inserts with cast-applied hydrogel membranes. The breakthrough times ofcalcium ions increased monotonically with increasing molefraction of TEGDA up to 9 mol%, Fig. 6. One-way ANOVAconfirmed the presence of a significant difference in meantime-lag as a function of crosslink density (p<0.0001). Themean time-lag for 1 mol%, 3 mol% and 5 mol% TEGDAinserts was found to be statistically similar. Hydrogel coatedinserts of 7 mol% and 9 mol% TEGDA had mean time lagvalues that were significantly different from 3 mol% andlower crosslink densities; hydrogel coated inserts of 12 mol% TEGDAwere only significantly different from 1 mol% andlower crosslink densities. The apparent diffusion coefficientdecreased exponentially as the TEGDAmole%was increased.Apparent diffusivity of 1 and 3mol% hydrogels were found tobe similar. All hydrogels above 3 mol% were significantlydifferent from 1 mol% hydrogels (p<0.006). Tortuosity of 1,3, and 5 mol% were similar, ranging from 1.7 to 4.5 and weresignificantly less than crosslink densities of ≥7 mol% whichrevealed tortuosities that ranged from 9.6 to 10.

3.2 Hydrogel characterization

Poly(HEMA)-based hydrogel attachment to the polycarbon-ate membranes did not require any surface modifications orpre-treatments (plasma, corona or oxidative wet chemical)to be performed. Preliminary works with other membranematerials such as poly(ethylene terephthalate) (PET fromBD Falcon and Corning) suggests that surface treatmentsmay be necessary. The first method by which this occurs isthrough photoinitiated surface grafting (Kato et al. 2003;Rivaton et al. 1983). The photoinitiator, DMPA, enters theexcited state by UV irradiation and creates surface radicalsupon the polycarbonate membrane through hydrogen ab-straction. The surface radicals may then undergo radicalchain polymerization with HEMA. Additionally, attachmentmay occur by nucleophilic addition reactions of the hydrox-yl groups of HEMAwith carbonate groups of polycarbonatein the presence of free radical initiators (Khan et al. 2005). Itwas expected that the freshly cast-applied and cross-linkedhydrogels would be all approximately the same thicknessdue to the viscosity of the monomer mixture having agoverning influence on the capillary action between thehydrophobic glass slide and insert membrane. Cast-appliedhydrogels showed little variance in as-cast dry film thick-ness. Hydrogel membranes used for as-cast thickness meas-urements were not subsequently used for experimentationunder conditions of hydration.

The use of n-BA at the mole fractions used in this report isthe most likely reason for the decreased total amount of im-bibed water, producing ca. 20–23% hydration for all hydrogelcrosslink densities at pH07.35. The changes in degree ofhydration and void fraction as a function of crosslink densityobserved in this work are practically invariant when comparedto previously used but similar compositions of hydrogels hav-ing degrees of hydration ranging from 30% to 50% for 1–12 mol% crosslink density (Boztas and Guiseppi-Elie 2009;Brahim et al. 2003). Invariability of these parameters is be-lieved to be due to strong hydrophobic interactions among n-BA and electrostatic interactions among the PC side chains

Table 2 Hydrogel thickness measurements, calcium ion breakthrough times, apparent diffusion coefficients, and tortuosity of calcium ions throughhydrogels as a function of varying cross-linking densities. 95% confidence intervals shown in parenthesis

Mol% TEGDA DiffusivityReplicates (n)

Mean hydratedthickness (μm)

Hydration (%) Void Fraction (%) Breakthroughtime (s)

Apparent DiffusionCoefficient, Dapp, ×10

−6

(cm2/s)

Tortuosity

1 mol% 3 30.6 (±0.3) 20.4 (±0.9) 19.8 (±4.4) 99 (±51) 1.40 (±0.07) 1.7 (±0.2)

3 mol% 4 30.7 (±0.3) 20.5 (±0.9) 24.1 (±3.3) 274 (±126) 0.72 (±0.05) 3.1 (±0.5)

5 mol% 6 31.3 (±0.1) 23.0 (±0.3) 24.5 (±2.7) 414 (±191) 0.54 (±0.02) 4.5 (±0.7)

7 mol% 6 30.8 (±0.2) 21.0 (±0.6) 23.6 (±0.7) 837 (±245) 0.22 (±0.01) 9.6 (±1.0)

9 mol% 3 31.1 (±0.2) 22.1 (±0.5) 26.5 (±1.9) 976 (±313) 0.17 (±0.01) 10.0 (±1.1)

12 mol% 3 31.2 (±0.2) 22.8 (±0.7) 27.5 (±2.4) 712 (±256) 0.18 (±0.01) 9.8 (±0.5)

Biomed Microdevices (2012) 14:549–558 555

creating virtual crosslinks that dominate the microstructure ofthe hydrogel compared to the composition of TEGDA.

The subject hydrogels have previously been shown todisplay elasticity that is highly dependent upon hydrationand swelling characteristics, which is directly proportional tocross-linking density (Boztas and Guiseppi-Elie 2009). It wasshown that the degree of hydration of hydrogels can becontrolled by varying cross linked density (Boztas andGuiseppi-Elie 2009); however, by the addition of highly hy-drophobic n-BA, the degree of hydration has been found to beessentially constant over all changes in covalent cross-linkingdensity. Correspondingly, the void fractions of the hydrogelshave remained constant, Fig. 7. Inclusion of n-BA served tobring the designed hydrogels into a more accessible rubberystate for softer mechanical properties in order to achieve agreater degree of interfacial and mechanical congruency withsurrounding tissues during in vivo implantation. Incorporationof a 10:3 HEMA:n-BA ratio decreased the glass transitiontemperature (Tg) from 110°C to approximately 80°C. Success-ful engineering of Tg through incorporation of n-BA into thenovel biomimetic hydrogel system has consequences of

decreased hydrophilicity and a fixed degree of hydrationirrespective of the crosslink density of the system.

3.3 Calcium ion diffusion and interaction

The trend in diffusivities are in concord with the effect of cross-link densities in previously studied and similarly composedbiomimetic hydrogels (Boztas and Guiseppi-Elie 2009). Thatis, diffusivity was high for low cross-link densities but fell to anasymptotic lower value with increasing cross-link density.Increasing cross-linking density of the hydrogels led to anobserved exponential decrease in apparent diffusivity whiletortuosity increased, Fig. 8. Qualitatively, tortuosity is expectedto increase with cross linked density. This was the case up toapproximately 7 mol% TEGDA hydrogels. At low crosslinkdensities ranging from 1mol% to 5mol%, tortuosity increased,leading to a steady decrease in diffusivity of calcium. Between7 mol% and 12 mol% TEGDA, the apparent diffusion coeffi-cient of calcium ions level off. This is due to the crosslinkdensity’s influence dropping exponentially. Other findingshave shown that increasing the continuous and hydrateddomains of a hydrogel will increase permeability to diffusingspecies (Boztas and Guiseppi-Elie 2009; El-Awady 2004; Itoet al. 2011; Murphy et al. 1988; Papadokostaki and Herouvim2002). Because hydration is maintained constant for all cross-link densities, tortuosity is the sole modality controlling thediffusivity for the examined hydrogel compositions.

The diffusion coefficient of free calcium ions in buffersolution has been reported to be 2.0–2.5×10−6 cm2/s (G. D.Smith et al. 2001; M. Naraghi and Neher 1997). The appar-ent diffusivity of calcium ions through a pure poly(HEMA)hydrogel of approximately 0.70 mol% crosslinking densityat 37°C has been observed to be about 1.85×10−6 cm2/s at aconcentration of 0.25 M calcium ions (Hamilton et al.

0

200

400

600

800

1000

1200

1400

1mol% 3mol% 5mol% 7mol% 9mol% 12mol%

Tim

e la

g (s

)

Hydrogel mol% TEGDA

Fig. 6 Calcium breakthrough times as a function of mole % TEGDA(cross-linking density) of hydrated hydrogels with 95% confidenceintervals (* indicates significant difference from hydrogels of 1 mol%to 3 mol%. † indicates significant difference from 1 mol%)

0%

5%

10%

15%

20%

25%

0.0%

10.0%

20.0%

30.0%

40.0%

50.0%

60.0%

70.0%

0% 2% 4% 6% 8% 10% 12% 14%

Voi

d F

ract

ion

TEGDA Mol%

ε Degree of Hydration

Deg

ree

of H

ydra

tion

%

Fig. 7 Apparent diffusivity and tortuosity as a function of mole %TEGDA (cross-linking density) with 95% confidence intervals

y = 1.3506e-19.97x

R² = 0.8792

1

3

5

7

9

11

13

0.0

0.5

1.0

1.5

2.0

2.5

0% 5% 10% 15%

Dap

px1

06 (cm

2 /s)

TEGDA

τ

Solution Diffusivity

Fig. 8 Void fraction and degree of hydration as a function of hydrogelcross-linking density with 95% confidence intervals (* indicates sig-nificant difference from hydrogels of 1 to 3 mol%. † indicates signif-icant difference from 1 mol%)

556 Biomed Microdevices (2012) 14:549–558

1988). The observed diffusivity of the currently formulated1.0 mol% TEGDA cross-linked hydrogel (1.40×10−6 cm2/s)is thus reasonable and consistent with reported values. Thisshows that none of the hydrogel compositions approximateto that of pure buffer.

Since it is well understood that ions permeate porouspolymers via the free water phase, the transport modelshould take into account the hydrodynamic radius of thediffusing species and its molecular architecture in relation-ship with the hydrogel matrix (Hamilton et al. 1988). Theoverall decrease in free water due to inclusion of n-BAwould limit calcium ion diffusivity through the hydrogelmatrix as fewer solvated calcium ions will be able to enterand move through the polymer matrix. The use of theHMMA constituent increases the hydrophilicity of thehydrogels compared to pure poly(HEMA). This may subse-quently increase the bound vs. free water character of thehydrogel matrix, which is known to affect partitioning anddiffusion of solutes through the hydrogel (Hoffman 2002).

Hydrogel matrix interactions with the diffusing species mayplay a prominent role as well. Interactions of calcium ions withthe pendant charged moieties of MPC, with an estimatedpKa109.2 and pKa2012.2, and DMAEMA (pKa07.3–7.5)(Brahim et al. 2003; D. Fournier et al. 2007) within the hydro-gels may be the source of electrostatic repulsion of the posi-tively charged calcium ions(N. Fatin-Rouge et al. 2003).However, the PC moiety of MPC is not sensitive to externalionic strength or pH influences as between pH 3 and pH 10 thephosphate anion and trimethylammonium cation yield an es-sentially neutral inner salt. The experiments were performed atapproximately 14–15 h of hydration. It has been shown thatMPC tends to migrate to the surface of the hydrogel, a processrequiring approximately 72–120 h (Abraham et al. 2005). Theresulting surface charge may contribute a surface membranepotential that influences the observed diffusivity.

4 Conclusions

A very simple two-compartment cell based on the use of cellculture inserts for the study of transport across hydrogel mem-branes has been described. These transport cells have beenused to study the effect of cross link density on the transportproperties of calcium ions across poly(HEMA)-based biomi-metic hydrogels possessing zwitterionic MPC and hydropho-bic n-BA constituents. The simple two-compartment transportcell based on the use of cell-culture inserts allows highthroughput transport studies in 6, 12 or 24-well microtiterplates. Diffusivity of calcium ions was observed to be affectedprimarily by the tortuosity of the hydrogels as well as poten-tially by ionic interactions of calcium with the polymer matrix.Tortuosity increases with increasing crosslink density, leadingto a trend of decreasing diffusivity of calcium ions. Inclusion of

n-BA into hydrogel compositions resulted in the paradoxicalalmost constant degree of hydration with increasing crosslinkdensity. Careful consideration must thus be given to the meansby which glass transition temperature is controlled in order toattain desired mechanical properties and diffusivity of mole-cules of interest. Fully hydrated biomimetic hydrogels do notapproximate to a pure water media for permeating calciumions. This may be due to a combination of electrostatic inter-actions with biomimetic moieties such as MPC and increasedfrictional drag upon diffusing ions by bound water within thehydrogel matrix. An order of magnitude change in calcium iondiffusivity within the subject hydrated hydrogels can be ac-quired by a change in crosslink density from 1.0 mol% to12.0 mol% while the degree of hydration is held constant,21.6±1.0%. Additional work that targets variation in theMPC content to probe the possible role of the inner salt anduses pH dependence to probe electrostatic interactions with theDMAEMA component is suggested.

Acknowledgements The authors acknowledge support from the USDepartment of Defense (DoDPRMRP) grant PR023081/DAMD17-03-1-0172 and the Consortium of the Clemson University Center forBioelectronics, Biosensors and Biochips (C3B). A.M. Wilsonacknowledges support from the Department of Chemistry, Universityof the West Indies, St. Augustine and ABTECH Scientific, Inc.

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