Calcium Concentration Effects on the Mechanical and BiochemicalProperties of Chondrocyte-Alginate Constructs

LEO Q. WAN,1 JIE JIANG,2 DIANA E. ARNOLD,1 X. EDWARD GUO,3 HELEN H. LU,2 and VAN C. MOW1

1The Liu Ping Laboratory for Functional Tissue Engineering Research, Department of Biomedical Engineering, ColumbiaUniversity, 351 Engineering Terrace, 500 West 120th Street, New York, NY 10027, USA; 2Biomaterials and Interface TissueEngineering Research, Department of Biomedical Engineering, Columbia University, New York, NY 10027, USA; and 3Bone

Bioengineering Laboratory, Department of Biomedical Engineering, Columbia University, New York, NY 10027, USA

(Received 17 January 2008; accepted 5 March 2008; published online 21 March 2008)

AbstractAlginate gel crosslinked by calcium ions (Ca2+)has been widely used in cartilage tissue engineering. How-ever, most studies have been largely performed in vitro inmedium with a calcium concentration ([Ca2+]) of 1.8 mM,while the calcium level in the synovial uid of the humanknee joints, for example, has been reported to be 4 mM oreven higher. To simulate the synovial environment, the twostudies in this paper were designed to investigate how thealginate scaffold alone, as well as the chondrocytes seededalginate gel responds to variations in medium [Ca2+]. InStudy A, the mechanical properties of 2% alginate hydrogelwere tested in 0.15 M NaCl and various [Ca2+] (1.0, 1.8, and4 mM). In Study B, primary bovine chondrocytes were seededin alginate gel, and biochemical contents and mechanicalproperties were determined after incubation for 28 days inthree [Ca2+] (1.8, 4, and 8 mM). For both studies, it wasfound that the magnitude of the complex shear modulus(|G*|) at 1 Hz doubled and the corresponding phase angleshift angle (d) increased >2 as a result of the approximate4-fold change in [Ca2+]. At high [Ca2+], the chondrocyteglycosaminogylcan (GAG) production inside the chondrocyte-alginate constructs was suppressed signicantly. This is likelydue to a decrease in the porosity of the chondrocyte-alginateconstructs as a result of compaction in structure caused by anincreased crosslinking density with [Ca2+]. These may beimportant considerations in the eventual successful imple-mentation of cartilage tissue-engineered constructs in theclinical setting.

KeywordsCartilage tissue engineering, Alginate gel, Chon-

drocytes, Mechanical property, Calcium ion.

INTRODUCTION

Articular cartilage is a dense white connective tissuecovering the moving articular ends of diarthrodialjoints.32 With traumatic injuries and progressive

degenerative diseases such as osteoarthritis (OA), somearticulating joint cartilage loses its essential mechanicalfunctions including load-supporting, wear and frictionminimization.26 Furthermore, since the time ofHippocrates (460BC), it has been known that articularcartilage has limited ability to repair itself, and thisobservation has been repeatedly demonstrated overtime.20 Today, autologous chondrocytes implantation(ACI)6 is the only cell-based therapy for cartilage repairapproved by US Food and Drug Administration(FDA). Because of the associated donor-site morbidityand prolonged cell cultural and surgical time, analternative approach has been proposed with the goalaimed to make reliable and functional substitutes forbiological tissues by applying the knowledge andprinciples of engineering and sciences.7,16,24 Alginategel crosslinked with calcium ions (Ca2+) has also beenwidely used for tissue engineering studies [e.g., 28,35,45]due to its documented biocompatibility, low toxicity,relatively low cost, and spontaneous gelation. Physio-logically, calcium concentration ([Ca2+]) plays animportant role in the function and organization ofarticular cartilage. These ions bind to proteoglycans byelectrostatic force to maintain structure and function ofthe tissue.8 Thus calcium metabolism and the changesin calcium ion concentration are important during thecartilage development and under pathological condi-tions.23 In particular, the calcium level in synovial uidof human knee joints has been reported to be 4 mM orhigher,27 while (inexplicably) most previous studieshave been performed in culture medium with a [Ca2+]of 1.8 mM. The increase in [Ca2+] is expected to changethe structure and mechanical properties of alginatehydrogels, and therefore affect the overall cellularresponse of embedded chondrocytes in such tissueengineered cartilage constructs in vivo. Within thein vivo intra-articular situation, these constructs are sub-jected to very high loading conditions, and as such, the

Address correspondence to Van C. Mow, The Liu Ping Labo-

ratory for Functional Tissue Engineering Research, Department of

Biomedical Engineering, Columbia University, 351 Engineering

Terrace, 500 West 120th Street, New York, NY 10027, USA.

Electronic mail: vcm1@columbia.edu

Cellular and Molecular Bioengineering, Vol. 1, No. 1, March 2008 ( 2008) pp. 93102DOI: 10.1007/s12195-008-0014-x

1865-5025/08/0300-0093/0 2008 Biomedical Engineering Society

93

mechanical properties of the hydrogel will have signif-icant inuence on the states stress and strain of theembedded chondrocytes will sustain.16,32

Alginate is a naturally derived linear polysaccharidecomprised of (14)-linked b-D-mannuronic acid anda-L-guluronic acid. Divalent cations, such as Ca2+, canbind the guluronic residues in adjacent alginate chainsto form an egg-box structure14,39 and cause the gela-tion of aqueous alginate solutions (Fig. 1). Mono-valent ions such as Na+ can also compete with calciumions for junction sites of guluronic residues andthereby weakening the alginate gel.25,37 The calciumcrosslinks can be readily dissolved in the absence of thedivalent ions, leading the gel to depolymerize. Alginatehydrogel has been utilized for cartilage repair, ACI andcartilage tissue engineering,18,35,45,46 and has beenreported to facilitate maintenance of the chondrocyticphenotype.19 Within the three-dimensional alginatematrix, the bone-marrow-derived stem cells have beenreported to differentiate into chondrocytes in an in vivorabbit study,9 and embedding dedifferentiated chon-drocytes in alginate hydrogel has led to the establish-ment of the characteristics of chondrocyte phenotype.4

Scaold mechanical properties are important forcartilage tissue engineering studies. Chondrocytes see-ded in scaolds are often stimulated with static or low-frequency dynamic compressive loading (leading tointerstitial perfusion which is important for the trans-port of nutrients and waste products), and oscillatoryshear to increase or accelerate tissue regeneration byincreasing cell biosynthetic activities [e.g.,15,43]. Thegenerated mechano-electrochemical signals are highlydependent on the composition of the matrix (scaffoldmaterials, and chondrocyte produced extracellularmatrix proteins, i.e., collagen, proteoglycan, etc.)mechanical properties such as compressive modulus,shear modulus, and permeability of the solid, porous-permeable matrix.32 Therefore, it is important to notonly quantify the mechanical properties of these con-structs and scaffolds, but to do so under physiological

ionic conditions.34 Most of the previously reportedmechanical properties of the alginate hydrogel havebeen obtained in various non-physiological conditionsat low-osmotic environment.31,37 therefore, are notdirectly applicable to tissue engineering for in vivoapplications. LeRoux et al.25 was the rst to study howthe mechanical properties of alginate hydrogel changewith the concentration of alginate and storage time ofthe hydrogel under a physiologically relevant ioniccondition (0.15 M NaCl and 1.8 mM CaCl2).Although it has been observed that alginate hydrogelmechanical properties highly depend on [Ca2+] in thegelation solution,41,42 no precisely controlled torsionalshear and compressional tests have been performed toquantify the effects of this [Ca2+] variation on the truematerial properties of alginate hydrogel under physi-ological ionic conditions, which has a [Ca2+] of4.0 mM or higher.1 It is not known how alginateresponds in these levels of [Ca2+], and more impor-tantly, how changes in [Ca2+] within the interstitiumof an alginate hydrogel could affect chondrocytebiosynthesis and matrix production in engineeredcartilage based on the use of such alginate constructs.The objective of this study is to investigate how

alginate hydrogel structure and mechanical propertieschange with medium calcium concentration and howchondrocytes embedded in alginate hydrogel respondto the variation in external calcium concentration inculture. To this end, two experiments were conducted:Study Aexamine the changes in mechanical proper-ties of alginate hydrogel in concentrations as afunction of [Ca2+] (1.0, 1.8, and 4 mM); StudyBdetermine the chemical contents and mechanicalproperties of chondrocyte-embedded alginate hydrogelafter culturing in media with in various calcium con-centrations (1.8, 4, and 8 mM) for 28 days.

FIGURE 1. Gelation of alginate solution with the addition of calcium or magnesium ions and formation of Egg box structure.

1Only a torsional shear test of cylindrical specimens, Fig. 2, provides

an equivoluminal deformation (i.e., no matrix dilatation) and hence

no motive forces to drive interstitial uid ow.47

WAN et al.94

METHODS

Sample Preparation and Cell Culture

Low-viscosity sodium salt alginic acid (A2158,Sigma, St Louis, MO) was used in these studies. Thisalginate is derived from Macrocystitis pyrifera, has anM/G ratio of 1.67 and a molecular weight of around50,000 Dalton. For Macrocystitis pyrifera, the molarfraction of guluronic acid is approximately 0.39 withan average block length of 5.0.25,39 Two percent (2%)alginate solutions were made and sterile ltered(0.22 lm porous size).In Study A, 2% alginate solutions were gelled by

diusion in 50 mM CaCl2 solution in a custom-madecylindrical Delrin mold (10 mm diameter 1.6 mmthickness). After 1.5 h, the samples (n = 4) wereremoved from the mold, and put into a solution with0.15 M NaCl and various concentrations of CaCl2: 1,1.84 mM. Media were exchanged regularly to keepthe external ion concentrations outside the samples asprescribed. The samples were immersed in the solu-tions for 1520 h to render them structurally stableand at diffusion equilibrium before performingmechanical tests.25

In Study B, chondrocytes were obtained from full-thickness articular cartilage of carpometacarpal jointsof skeletally immature, 14 week-old calves from alocal abattoir (Rutland, VT) following enzymaticdigestion described by Jiang et al.22 Briey, articularcartilage was minced into small pieces and was thensubjected to serial enzymatic digestion with 0.25%pronase (Calbiochem, San Diego, CA) for 1 h and0.05% collagenase (Sigma) for 4 h at 37 C. Theresulting cell suspension was ltered through a sterilemembrane with a pore size of ~30 lm and centrifugedfor 5 min. The cell pellet was then mixed with 2%alginate solution at a density of 30 million cells/mL.The cell-alginate mixture was gelled in a custom moldwith 50 mM CaCl2 in phosphate buffered saline (PBS)for 30 min before the chondrocyte-alginate constructswere removed from the mold. These samples were thenincubated at 37 C and 5% CO2 for 28 days inDulbeccos modied Eagles medium (DMEM) sup-plemented with 10% fetal bovine serum (FBS, Cellgro-Mediatech), 1% non-essential amino acids, 100 units/mLpenicillin, 100 lg/mL streptomycin, and 20 lg/mLascorbic acid (Sigma). Concentrated CaCl2 solution(2 M) was added to reach calcium levels of 1.8, 4, and8 mM. During incubation, media were changed everyother day.

Mechanical Testing

The diameter (d) of each sample from both Study Aand Study B was measured with a stereomicroscope

(model PP&E 56939; Bausch and Lomb, Rochester,NY). The mechanical tests were performed on ashearstrain controlled rheometer (ARES-LS1, TAinstruments, New Castle, DE). The sample was placedbetween two at-rough porous platens (rms surfaceroughness is 30 lm) to prevent it from slippingagainst the specimen, and immersed, to preventdehydration, in the same solution for Study A andstorage medium at 37 C for Study B47 (Fig. 2). First,a normal tare load of about 0.025 N was adminis-tered, and the reference thickness h of the sample wasdetermined from the axial position readings of therheometer. After 5 min of compressive-stress relaxa-tion, the normal (i.e., vertical) force component (F1)was recorded. A 15% (e) normal strain was thenapplied, and the normal force (F2) was again deter-mined after another 25 min. From this normal force,the equilibrium compressive modulus (Eeq) of thisspecimen was calculated at 15% compressive strain asfollows:

Eeq re where r F2 F1pd 2=4

: 1

For Study A, a step shear stress relaxation test wasperformed after 15% compression, a step shear strainof 0.01 (c) radian was administered by applying anangular displacement h = 2 ch/d (see Fig. 2) and fol-lowed with a 40-min shearstress relaxation. Theequilibrium shear moduli (Geq) were then calculatedfrom the equilibrium torque applied (Teq) to the sam-ple with the formula below:

Geq Teqd2Ipc

; 2

where Ip is the polar moment of inertia of a cylinderand given by Ip = pd

4/32.For both Studies A and B, a dynamic shear test was

performed over frequencies ranging from 0.01 to 10 Hzon a logarithmic frequency sweep with shear strainamplitude c0 = 0.01 radian. The complex (dynamic)shear modulus was calculated from G* = Td/(2Ipc) asa function of frequency (x), where, c is a sinusoidalshear strain applied on the sample with the formc = c0sin(xt), where c0 is the amplitude and x is theangular frequency, and T is the torque response.Generally, G* is a complex number for viscoelasticmaterials such as hydrogels, and can be expressed as,where G* = G + iG, G and G are the storagemodulus and loss modulus, respectively. The magni-tude of the dynamic shear modulus (|G*|) is given by

jGj

G02 G002q

; and the phase shift angle (d)

between the applied strain and the torque response iscalculated from d = tan-1(G/G).47

Calcium Concentration Effects on the Mechanical and Biochemical Properties 95

Biochemical Assays and Histology

For Study B, after mechanical tests, each samplewas rst washed with PBS and then halved with onehalf frozen for biochemical assays and the other halfused for histology. For biochemical assays, the samplewas rst gently blotted dry and weighed (wet weight),and then the sample was lyophilized and the dry weightwas obtained. Water content (%) was calculated as(wet weightdry weight)/wet weight. Samples werethen digested for 16 h at 60 C with 20 lL/mL papainin 0.1 M sodium acetate, 10 mM cysteine HCl, and50 mM ethylenediaminetetraacetate (EDTA).An aliquot of the digest was used to determine the

total DNA per sample using the PicoGreen dsDNAassay30 (Molecular Probes, Eugene, OR) following theprotocol provided by the manufacturer. Fluorescencewas measured with a microplate reader (Tecan,Maennedorf, Switzerland) with excitation and emis-sion wave-lengths of 485 and 535 nm, respectively.To quantify the glycosaminogylcan (GAG) content,

a modied 1,9-dimethylmethylene blue (DMMB) dye-binding assay10,11 with chondroitin-6-sulfate (Sigma)as a standard was used. To account for the anionicnature of the carboxyl groups on the alginate hydrogel,the pH of the DMMB dye was adjusted to be 1.5 withthe concentrated formic acid (Sigma) so that only thesulfated GAG-DMB complexes were detectable withthe spectrophotometer. The absorbance at both 540and 595 nm was used to improve signal detection.38

Sample collagen content was quantied with asimplied hydroxyproline assay.21,36,40 Specically, analiquot of the digest was hydrolyzed with high con-centration sodium hydroxide (2 M) at 120 C for20 min. The hydrolyzate was then oxidized by a buf-fered choloramine-T (Sigma) reagent for 25 min beforethe addition of Ehrlichs reagent (15% p-dimethyl-aminobenzaldehyde in the mixed solution of isopro-panol/percholoric acid (2:1)), and the absorbance wasmeasured at 550 nm (Tecan). The hydroxyprolinecontent was converted to collagen content using aconversion ratio of 1:10 hydroxyproline: collagen.44

For histology, the sample was xed with acid for-malin (4% formaldehyde in 70% ethanol and 5%acetic acid), dehydrated with a graded series of etha-nol, and embedded in paran for histological study.The samples were sectioned (7.5 lm) and mounted onmicroscope slides prior to staining. Sample GAG dis-tribution was stained with Alcian blue staining atpH = 1.5, and collagen distribution was ascertainedwith Picosirius red staining.3 Stained sections wereimaged using the Axiovert 35 microscope (Zeiss,Oberkochen, Germany).

Statistical Analysis

Data are presented as means standard devia-tions. In Study A, one-way analysis of variance(ANOVA) was performed on the equilibrium

FIGURE 2. Schematic illustration of an alginate gel disk in the torsional shear and compression testing device (ARES-LS1, TAinstruments).

WAN et al.96

compressive modulus, shear modulus, and the magni-tude of dynamic shear modulus and phase shift angleat 1 Hz to assess the eects of varying calcium con-centrations on alginate hydrogel. In study B, ANOVAwas also performed on chemical contents for thechondrocyte-embedded alginate hydrogel constructs.For both studies, the eects of external ion concen-trations and frequency on the magnitude of dynamicshear modulus |G*| and phase shift angle d wereassessed by two-way ANOVA, and the effects of thefrequency on |G*| at the lowest frequency (0.01 Hz)and the highest frequency (10 Hz) were tested with anANOVA with repeated measures. StudentNewmanKeuls (SNK) tests were performed at a 95% condenceinterval to determine specic differences betweengroups. All statistical analyses were performed usingthe Statistical Analysis Software (SAS, Cary, NC).

RESULTS

Eects of [Ca2+] on Alginate Hydrogel

For alginate hydrogel samples, it was found that theequilibrium shear modulus Geq depended signicantlyon the calcium concentration in the external solutions(p0.05; Fig. 3a). The values for Geq more thandoubled (1.26 0.28 kPa vs. 2.72 0.11 kPa) when[Ca2+] increased from 1 to 4 mM.Similar to the equilibrium shear behavior, both the

magnitude of the dynamic shear modulus and phaseshift angle at 1 Hz increased signicantly with calciumconcentration from 1 to 4 mM (p

more deposition at the exteriors of the constructs thanthe interiors and that the deposition in the interiorsdecreases with increasing [Ca2+]. Two important differ-ences are noted between these chondrocyte-embeddedalginate hydrogels tissue engineering constructs andnative articular cartilage: (1) the water content of these

tissue engineering constructs are very high, i.e., >93%,whereas in normal articular cartilage it is ~78%1; and(2) the collagen does not appear to be organized in alayered-anisotropic manner as in native articularcartilage.32

DISCUSSION

The objective of this study was to investigate theeects of the physiologic synovial level [Ca2+] on

0

20

40

60

80

1.8mM 4mM 8mM

Mod

ulus

(kPa

)

0

4

8

12

16*

**

Calcium Concentration

Eeq|G*|

*

**

(a)

0

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60

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100

0.01 0.1 1 10

Mag

nitu

de o

fD

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hear

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(KPa

)

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*

*

Frequency (Hz)

1.8mM 4mM 8mM *

*

*

(b)

Phase shift angle

()

FIGURE 4. (a) Effects of calcium concentration on the equi-librium compressive modulus Eeq and the magnitude ofdynamic shear modulus |G*| and phase shift angle d at 1 Hz forchondrocyte-embedded alginate hydrogel constructs afterincubation for 28 days (n = 4; *Significant differencesbetween all groups, **Significantly different from groups[Ca2+] = 1.8 and 4 mM; p < 0.05, SNK); (b) Effects of calciumconcentration on frequency-dependent dynamic shear mod-ulus of chondrocyte-alginate constructs after incubation for28 days. (*Significantly different from 0.01 Hz; p < 0.05, SNK).

TABLE 1. Chemical contents (total weights) and dimension of chondrocyte-alginateconstructs (n = 4) after incubation for 28 days.

[Ca2+] (mM)

1.8 4.0 8.0

Diameter (mm) 10.79 0.38 10.86 0.51 9.53 0.12*

Wet weight (mg) 302 10 286 2 236 3*

Water (mg) 287 9 269 2 221 2*

Water content (%) 95.1 0.4 94.4 0.4 93.7 0.4

DNA (lg) 25.85 5.54 29.35 7.23 22.21 9.71Collagen (mg) 2.29 0.73 1.89 0.10 1.61 0.11

GAG (mg) 4.17 0.73 4.09 0.15 2.49 0.35*

* Significantly different from [Ca2+] = 1.8 mM and [Ca2+] = 4.0 mM; p < 0.05, SNK.

0

2

4

6

1.8 4 8

Chem

ical

Con

tent

(mg/d

isk)

GAG Collagen

*

Calcium Concentration (mM)

*

(a)

(b)

FIGURE 5. Effects of calcium concentration (a) on glycos-aminoglycan (GAG) and collagen contents inside chondrocyte-embedded alginate hydrogel constructs after incubation for28 days. (n = 4; *Significant differences relative to [Ca2+] = 1.8and 4.0 mM; p < 0.05, SNK) and (b) on the distribution of GAGand collagen inside constructs (Top: Alcian Blue staining;Bottom: Picosirius Red staining).

WAN et al.98

alginate hydrogel and alginate hydrogel seeded withchondrocytes. Specically, compression and shearproperties of the samples were determined as a func-tion of [Ca2+]. In addition, the effects of the [Ca2+]increase on matrix synthesis and distribution bychondrocytes in alginate hydrogel were investigated.Overall, it was found that changes in Ca2+ concen-tration signicantly altered the shear properties ofalginate hydrogel and chondrocyte-embedded alginatehydrogel constructs under both the equilibrium anddynamic tests, while surprisingly no signicant changesin the compressive properties were detected. This hadoccurred even with the modication of the samplesswelling pressure (Donnan osmotic pressure). Thephase shift angle of dynamic shear tests, a measure ofthe matrix viscoelasticity, was found to increase sig-nicantly with calcium concentration. The GAG con-tent for chondrocyte-embedded alginate hydrogelconstructs decreased signicantly with calcium con-centration, while their shear properties increased. Thevariation of scaffold shear properties with calciumconcentration could signicantly alter the magnitudeof mechano-electrochemical signals in situ (such asstresses, strains, uid ow and electrical potential, i.e.,with a decrease of the charged GAG molecules) andtherefore the chondrocyte biosynthetic response,and also affect the continued integrity of the constructsand the stress-strains elds surrounding of host carti-lage tissue under joint loading.32

Hydrogels have often been modeled as biphasicmaterials consisting of a mixture of two incompressiblephases: solid phase and water phase.13,29 Hydrogelsexhibit viscoelastic behavior when the water or uidinside the porous-permeable hydrogel is forced togradually ow out under compression. Previously, thelinear isotropic biphasic model33 has been used todescribe the mechanical behavior of agarose and studynutrient transport through the pores of an agarosematrix.13,29 For this study on the effects of [Ca2+] onalginate hydrogel, however, the situation is much morecomplex because the hydrogel matrix is negativelycharged due to the carboxyl groups present on gulu-ronic and mannuronic residues at the physiological pHrange and mechano-electrochemical effects may not beignored.32 Similar to articular cartilage, the alginatehydrogel can be, and has been successfully, treated as atriphasic material which includes an additional phase,mobile ions owing through a charged, porous-permeable matrix. Due to the existence of xed nega-tive charges attached to the solid matrix of alginatehydrogel, a Donnan osmotic pressure will be generatedwithin the hydrogel, and complex mechano-electrochemical events will occur inside the hydrogelupon mechanical or osmotic loading.27,32 In this con-text, the torsional shear test has special advantages in

its ability to determine the shear properties of thespecimen since it is an equivoluminal test and thusinterstitial uid ow is minimized. No uid ow rela-tive to the solid matrix occurs when the applied shearstrain is kept small during the test. Therefore, unlikethe compressive response, the viscoelastic behaviorunder torsional shear tests comes solely from the solidmatrix of the hydrogel. In other words, the pure sheartest is particularly advantageous for quantifying theviscoelastic behavior of the solid phase of alginatehydrogel.For the alginate hydrogel, the measured equilibrium

compressive modulus (~4 kPa) and shear modulus(~2 kPa) at blood plasma ionic condition (0.15 MNaCl and 1.8 mM CaCl2) agree well with previousexperimental results obtained by LeRoux et al.25 Inthis study, the extent to which the mechanical prop-erties change under physiological ionic conditions hasbeen quantied as well, and the results indicate thatvariations in calcium concentration may signicantlyaffect the shear properties of alginate hydrogel.Increased calcium concentration will promote theformation of additional crosslinks inside the alginatehydrogel and stiffen the alginate samples. Intuitively,the phase shift angle would accordingly decrease.However, the measured phase shift angle was shown tosignicantly increase with [Ca2+] (d: 1.2 0.6 vs.3.4 0.4, at [Ca2+] = 1.0 mM vs. 4.0 mM).This might be due to the increase in solid volumefraction as a result of the sample shrinkage (swollendiameter: 9.67 0.06 mm vs. 9.19 0.07 mm, at[Ca2+] = 1.0 mM vs. 4.0 mM). It led to higher fric-tion between polymer chains inside the alginatehydrogel which dominated the effect of increased Ca2+

cross-linking density, resulting in the increase of thephase shift angle.25

For chondrocyte-embedded alginate hydrogel con-structs, mechanical properties increase with time as aresult of the longitudinal cellular production of extra-celluar matrix collagen and GAG. The compressiveproperties of these composite constructs in the mediumwith a [Ca2+] of 1.8 mM at Day 28 are comparable tothose reported in several previous studies.29,46 Awadet al.2 measured shear properties of alginate tissueengineered constructs using human adipose-derivedstem cells and focused on the potential of these cells forchondrogenic differentiation. However, no shearproperties of alginate constructs seeded with primarychondrocytes have been reported.From Day 1 to Day 28, the equilibrium compressive

modulus increased about 5 folds from 4 to 18 kPa,while the magnitude of dynamic shear modulus at 1 Hzincreased about 12 folds from 3 to 36 kPa. Thisincrease in both compressive and shear properties withtime is directly associated with the deposition of the

Calcium Concentration Effects on the Mechanical and Biochemical Properties 99

GAG and collagen inside the constructs and theresulting decrease in the water content or matrixporosity. The dependence of mechanical properties onthe hydrogel porosity has been reported for both softtissue such as articular cartilage1,32 and hard tissuesuch as bone.17 Furthermore, the phase shift angle at1 Hz increased from 2 to 9 from Day 1 to Day 28,which indicates the internal energy dissipation increasemay be the result of the increase in cell matrix depo-sition with time. Previously, it was reported that thephase shift angle of alginate hydrogel remains essen-tially unchanged when the alginate concentrationvaried as a result of the counteracting effects from theincrease of calcium crosslinks and the solid content.25

The calculated increase in phase shift angle suggeststhat in the chondrocyte-embedded alginate hydrogels,the deposited GAG and collagen have yet to formeffective crosslinks as strong as the linkages betweenguluronic residues of the alginate chains.47

When compared to the native articular cartilage, theequilibrium mechanical properties of these cell-seededconstructs are relatively low even after 28 days ofincubation. Specically, the compressive modulus ofthese chondrocyte-laden alginate constructs is about25 times lower than that of native cartilage, while thedynamic shear modulus is about 30 times lower.32 Thisis largely due to the relatively lower chemical contentsin comparison with native cartilage, with the GAGcontent of the constructs being 3 times lower, and thecollagen content approximately 10 times lower. Thismay be due to their very high water content (>93%)when compared to normal native articular cartilage(~78%). It is known that for osteoarthritic cartilage,the % hydration increases as the histochemical mea-sures increase1,27,32; when the hydration of such tissuesreaches >90%, the tissue no longer can provide loadsupport. For the native cartilage, the phase shift angleat 1 Hz is about 10,47 while it is around 9 for chon-drocyte-embedded alginate hydrogel constructs afterincubation for 28 days. For native articular cartilage,the shear property is highly dependent on the collagencontent,47 while for chondrocyte-alginate constructs itis dependent on the density of Ca2+ crosslinks, ratherthan the collagen content, as indicated by the increaseof dynamic shear modulus with increased [Ca2+]despite the decrease of the collagen and GAG contents.This difference is likely due to the small amount ofcollagen brils produced by chondrocytes in vitro, andits restricted deposition to a small region surroundingthe cells (Fig. 5b bottom). Consequently, collagenbers have not been well assembled nor well organizedwithin the alginate hydrogel to contribute to theincrease in shear modulus. The lack of a collagen ultra-structural architecture defeats the tension-compressionnonlinearity that is needed for effective interstitial uid

pressurization essential for hydrodynamic loadsupport.3234

In this study, no signicant eects of [Ca2+] onthe equilibrium compressive modulus have been de-tected for the alginate hydrogel and chondrocyte-embedded alginate hydrogel constructs, but the shearmodulus did increase signicantly with [Ca2+]. Theexact cause is not known and needs future investi-gation. It could be due to the bimodular response ofpolymer chains and collagen brils, as they exhibitmuch higher modulus in tension while lower modulusunder compression. The polymer chains and collagenbrils aligned in the tensile principal stress directionsignicantly contribute to the shear modulus of thesolid matrix,32 and therefore shear tests are moresensitive to the crosslinking density changes causedby variations of [Ca2+]. It could also be due tomatrix inhomogeneities inside the samples. As indi-cated by our histological staining (Fig. 5b), the highcalcium level group usually contains a thicker exte-rior or surface region with high GAG and collagencontents. The dense extracellular matrix there maysignicantly increase the shear modulus of the wholeconstructs while contributing little to the compressivemodulus.Generally, a calcium concentration larger than

10 mM is considered to be toxic to cells, but thedirect eects of calcium level below 10 mM onprimary chondrocytes have not been well-studied.Collagen production was reported to be the lowest at[Ca2+] = 1 to 2 mM in a chondrocyte-monolayerstudy, and an increase of [Ca2+] from 2 mM stimu-lated collagen production.23 Other studies show that alower calcium level can stimulate the Type X collagensynthesis and suppress the Type I collagen synthesiswhile leaving the Type II collagen unchanged.5,12 Nosignicant effect of calcium concentration on GAGproduction has been reported. Based on these studies,no signicant decrease of ECM (i.e., collagen andGAG) production has ever been reported when [Ca2+]increases from 1.8 to 8.0 mM. In contrast, we noticedin this study that both GAG and collagen in the his-tological staining of Study B (Fig. 5b) are present inlower amounts at 8 mM than 1.8 mM in the constructinterior, whereas their distribution are comparable forall groups at the rim of the samples. These observa-tions suggest that the variation in [Ca2+] has negligibledirect effects on chondrocyte synthetic response andthat the difference in matrix production is probablycaused by nutrient transport suppression at higher[Ca2+] due to increased hydrogel cross-linking density.Bioreactors coupled with perfusion and dynamicloading devices can be used to test this hypothesis, andpossibly to improve the nutrient transport throughthese tissue engineered constructs.

WAN et al.100

CONCLUSION

This study has provided new insight into the changesof the basic structure and viscoelastic properties of thealginate hydrogels subject to varying calcium concen-tration under physiologically relevant ionic conditionsas well as its inuence on the response of primarybovine chondrocytes seeded in this alginate scaold.These ndings have enhanced our understanding ofhow the outcome of alginate-based cartilage repairtherapies are aected by the elevated calcium level aswould be in the knee joints in both ACI and cartilagetissue engineering. It was found that moderateincreases in calcium concentration can signicantlyelevate the shear modulus of alginate hydrogel andchondrocyte-alginate constructs. The compaction instructure due to loss of total volume as a result ofincreased [Ca2+] decreases the porosity of the chon-drocyte-alginate constructs, which impedes nutrienttransport, resulting in lower ECM production and cellproliferation. These are important considerations inthe implementation of tissue-engineered constructs forcartilage repair.

ACKNOWLEDGMENTS

This study is supported by Whitaker FoundationSpecial Development Award (Mow), Stanley Dickerand Shelly Ping Liu endowments (Mow), NIH grantsR01 AR048287 and R21 AR052417 (Guo), and R21AR052402-01A1 (Lu). The authors thankDrs.GordanaVunjak-Novakovic and Jeremy J. Mao for their helpfulsuggestions and discussion of results.

REFERENCES

1Armstrong, C. G., and V. C. Mow. Variations in theintrinsic mechanical properties of human articular cartilagewith age, degeneration, and water content. J. Bone JointSurg. Am. 64:8894, 1982.2Awad, H. A., M. Q. Wickham, H. A. Leddy, J. M. Gimble,and F. Guilak. Chondrogenic differentiation of adipose-derived adult stem cells in agarose, alginate, and gelatinscaffolds. Biomaterials 25:32113222, 2004.3Bancroft, J. D., and M. Gamble. Theory and Practice ofHistological Techniques. New York: Churchill Living-stone, 2002.4Bonaventure, J., N. Kadhom, L. Cohen-Solal, K. H. Ng,J. Bourguignon, C. Lasselin, and P. Freisinger. Reexpres-sion of cartilage-specic genes by dedifferentiated humanarticular chondrocytes cultured in alginate beads. Exp. CellRes. 212:97104, 1994.5Bonen, D. K., and T. M. Schmid. Elevated extracellularcalcium concentrations induce type collagen synthesis inchondrocyte cultures. J. Cell Biol. 115:11711178, 1991.

6Brittberg, M., A. Lindahl, A. Nilsson, C. OhlssonO. Isaksson, and L. Peterson. Treatment of deep cartilagedefects in the knee with autologous chondrocyte trans-plantation. N. Engl. J. Med. 331:889895, 1994.7Butler, D. L., S. A. Goldstein, and F. Guilak. Functionaltissue engineering: the role of biomechanics. J. Biomech.Eng. 122:570575, 2000.8Campo, R. D. Effects of cations on cartilage structure:swelling of growth plate and degradation of proteoglycansinduced by chelators of divalent cations. Calcied TissueInt. 43:108121, 1988.9Diduch, D. R., L. C. Jordan, C.M.Mierisch, andG. Balian.Marrow stromal cells embedded in alginate for repair ofosteochondral defects. Arthroscopy 16:571577, 2000.

10Enobakhare, B. O., D. L. Bader, and D. A. Lee. Quanti-cation of sulfated glycosaminoglycans in chondrocyte/alginate cultures, by use of 1,9-dimethylmethylene blue.Anal. Biochem. 243:189191, 1996.

11Farndale, R. W., C. A. Sayers, and A. J. Barrett. A directspectrophotometric microassay for sulfated glycosamino-glycans in cartilage cultures. Connect. Tissue Res. 9:247248, 1982.

12Gigout, A., M. Jolicoeur, and M. D. Buschmann. Lowcalcium levels in serum-free media maintain chondrocytephenotype in monolayer culture and reduce chondrocyteaggregation in suspension culture. Osteoarth. Cartilage13:10121024, 2005.

13Goldsmith, A. A., and S. E. Clift. Investigation into thebiphasic properties of a hydrogel for use in a cushion formreplacement joint. J. Biomech. Eng. 120:362369, 1998.

14Grant, G. T., E. R. Morris, D. A. Rees, P. J. C. Smith, andD. Thom. Biological Interactions between polysaccharidesand divalent cationsegg-box model. Febs. Lett. 32:195198, 1973.

15Guilak, F., and C. T. Hung. Physical regulation ofcartilage metabolism. In: Basic Orthopaedic Biomechan-ics and Mechano-Biology, edited by V. C. Mow, andR. Huiskes. Philadelphia: Lippincott Williams andWilkins, 2005, pp. 259300.

16Guilak, F., and V. C. Mow. The biomechanical environ-ment of the chondrocyte: a biphasic nite element model ofcell-matrix interactions in articular cartilage. J. Biomech.33:16631673, 2000.

17Guo, X. E. Mechanical properties of cortical bone andcancelllous bone tissue. In: Bone Mechanics Handbook,edited by S. C. Cowin. Boca Raton, FL: CRC Press, 2001,pp. 1011.

18Guo, J. F., G. W. Jourdian, and D. K. MacCallum. Cultureand growth characteristics of chondrocytes encapsulated inalginate beads. Connect. Tissue Res. 19:277297, 1989.

19Hauselmann, H. J., R. J. Fernandes, S. S. Mok, T. M.Schmid, J. A. Block, M. B. Aydelotte, K. E. Kuettner, andE. J. Thonar. Phenotypic stability of bovine articularchondrocytes after long-term culture in alginate beads.J. Cell Sci. 107(Pt 1):1727, 1994.

20Hunziker, E. B. Articular cartilage repair: are the intrinsicbiological constraints undermining this process insupera-ble? Osteoarth. Cartilage 7:1528, 1999.

21Huszar, G., J. Maiocco, and F. Naftolin. Monitoring ofcollagen and collagen fragments in chromatography ofprotein mixtures. Anal. Biochem. 105:424429, 1980.

22Jiang, J., S. B. Nicoll, and H. H. Lu. Co-culture of osteo-blasts and chondrocytes modulates cellular differentiationin vitro. Biochem. Biophys. Res. Commun. 338:762770,2005.

Calcium Concentration Effects on the Mechanical and Biochemical Properties 101

23Koyano, Y., M. Hejna, J. Flechtenmacher, T. M. Schmid,E. J. Thonar, and J. Mollenhauer. Collagen and proteo-glycan production by bovine fetal and adult chondrocytesunder low levels of calcium and zinc ions. Connect. TissueRes 34:213225, 1996.

24Langer, R., and J. P. Vacanti. Tissue engineering. Science260:920926, 1993.

25LeRoux, M. A., F. Guilak, and L. A. Setton. Compressiveand shear properties of alginate gel: effects of sodium ionsand alginate concentration. J. Biomed. Mater. Res. 47:4653, 1999.

26Mankin, H. J., V. C. Mow, J. A. Buckwalter, J. P. Iannotti,and A. Ratcliffe. Articular cartilage structure, composition,and function. In: Orthopaedic Basic Science: Biology andBiomechanics of the Musculoskeletal System, edited byJ. A. Buckwalter, T. A. Einhorn, and S. R. Simon.Rosemont, IL: American Academy of Orthopaedic Sur-geons Publishers, 2000, pp. 443470.

27Maroudas, A. Physicochemical properties of articular car-tilage. In: Adult Articular Cartilage, edited by M. A. R.Freeman. Kent, UK: Pitman Medical, 1979, pp. 215290.

28Masuda, K., R. L. Sah, M. J. Hejna, and E. J. Thonar. Anovel two-step method for the formation of tissue-engineered cartilage by mature bovine chondrocytes: thealginate-recovered-chondrocyte (ARC) method. J. Orthop.Res. 21:139148, 2003.

29Mauck, R. L., M. A. Soltz, C. C. Wang, D. D. WongP. H. Chao, W. B. Valhmu, C. T. Hung, and G. A.Ateshian. Functional tissue engineering of articular car-tilage through dynamic loading of chondrocyte-seededagarose gels. J. Biomech. Eng. 122:252260, 2000.

30McGowan, K. B., M. S. Kurtis, L. M. Lottman, D.Watson,andR.L. Sah. Biochemical quantication ofDNA in humanarticular and septal cartilage using PicoGreen and Hoechst33258. Osteoarth. Cartilage 10:580587, 2002.

31Mitchell, J. R., and J. M. V. Blanshard. Rheologicalproperties of alginate gels. Rheol. Acta. 13:180184, 1974.

32Mow, V. C., W. Y. Gu, and F. H. Chen. Structure andfunction of articular cartilage and meniscus. In: BasicOrthopaedic Biomechanics and Mechano-Biology, editedby V. C. Mow, and R. Huiskes. Philadelphia: LippincottWilliams and Wilkins, 2005, pp. 181258.

33Mow, V. C., S. C. Kuei, W. M. Lai, and C. G. Armstrong.Biphasic creep and stress relaxation of articular cartilage incompression: theory and experiments. J. Biomech. Eng.102:7384, 1980.

34Mow, V. C., C. C. Wang, and C. T. Hung. The extracel-lular matrix, interstitial uid and ions as a mechanical

signal transducer in articular cartilage. Osteoarth. Cartilage7:4158, 1999.

35Paige, K. T., L. G. Cima, M. J. Yaremchuk, B. L. SchlooJ. P. Vacanti, and C. A. Vacanti. De novo cartilage genera-tion using calcium alginate-chondrocyte constructs. Plast.Reconstr. Surg. 97:168178, 1996: discussion 179180.

36Reddy, G. K., and C. S. Enwemeka. A simplied methodfor the analysis of hydroxyproline in biological tissues.Clin. Biochem. 29:225229, 1996.

37Segeren, A. J. M., J. V. Boskamp, and M. Vandentempel.Rheological and swelling properties of alginate gels. Fara-day Discuss. 1974:255262, 1975.

38Seibel, M. J., W. Macaulay, R. Jelsma, F. Saed-Nejad, andA. Ratcliffe. Antigenic properties of keratan sulfate: inu-ence of antigen structure, monoclonal antibodies, and anti-body valency. Arch. Biochem. Biophys. 296:410418, 1992.

39Smidsrod, O., and G. Skjak-Braek. Alginate as immobili-zation matrix for cells. Trends Biotechnol. 8:7178, 1990.

40Stegemann, H., and K. Stalder. Determination of hydroxy-proline. Clin. Chim. Acta. 18:267273, 1967.

41Thu, B., P. Bruheim, T. Espevik, O. Smidsrod, P. Soon-Shiong, and G. Skjak-Braek. Alginate polycation micro-capsules. I. Interaction between alginate and polycation.Biomaterials 17:10311040, 1996.

42Velings, N. M., and M. M. Mestdagh. Physicochemicalproperties of alginate gel beads. Polym. Gels Networks3:311330, 1995.

43Vunjak-Novakovic, G., and S. A. Goldstein. Biomechani-cal principles of cartilage and bone tissue engineering. In:Basic Orthopaedic Biomechanics and Mechano-Biology,edited by V. C. Mow, and R. Huiskes. Philadelphia:Lippincott Williams and Wilkins, 2005, pp. 343408.

44Vunjak-Novakovic, G., I. Martin, B. Obradovic, S. Treppo,A. J. Grodzinsky, R. Langer, and L. E. Freed. Bioreactorcultivation conditions modulate the composition andmechanical properties of tissue-engineered cartilage. J. Ort-hop. Res. 17:130138, 1999.

45Wong, M., M. Siegrist, V. Gaschen, Y. Park, W. Graber,and D. Studer. Collagen brillogenesis by chondrocytes inalginate. Tissue Eng. 8:979987, 2002.

46Wong, M., M. Siegrist, X. Wang, and E. Hunziker.Development of mechanically stable alginate/chondrocyteconstructs: effects of guluronic acid content and matrixsynthesis. J. Orthop. Res. 19:493499, 2001.

47Zhu, W., V. C. Mow, T. J. Koob, and D. R. Eyre. Visco-elastic shear properties of articular cartilage and the effectsof glycosidase treatments. J. Orthop. Res. 11:771781,1993.

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