Multi-Featured Macroporous Agarose–Alginate Cryogel: Synthesis and Characterization for Bioengineering Applications

Full Paper

22

Multi-Featured Macroporous Agarose–AlginateCryogel: Synthesis and Characterization forBioengineering Applications

Anuj Tripathi, Ashok Kumar*

In this study agarose–alginate scaffolds are synthesized using cryogelation technology indifferent formats like monolith, sheet, discs, and beads, and show amiable mechanicalstrength like soft tissue properties and high interconnected macroporous degradable archi-tecture. In cell–material interactions, fibroblast (NIH-3T3) cells showed good adherence andproliferation on these scaffolds presenting its potentialapplication in soft tissue engineering. The application ofcryogel beads and monoliths was also examined by theefficient immobilization of bacterial cells (BL21) on thesematrices revealing their use for recovery of product fromcontinuous fermentation systems without cell leakage.These scaffolds also showed potential as a filter forrepeated recovery of heavy metal binding, such as copperand nickel from the waste water. The cryogels preparedherein do have a number of unique features that makethem an important class of soft materials for developingmulti-featured scaffolds as a novel carrier for bioengi-neering applications.

Introduction

Hydrogels are a water insoluble, hydrophilic, three-dimen-

sional (3D)networkofcrosslinkedpolymerchains thathave

the capability of imbibing high water content or biological

fluids. Hydrogels have attracted a great deal of attention

and significant progress has been made in designing,

synthesizing, and using these materials for many bioengi-

neering applications. In addition, they have been widely

used in drug and protein delivery systems, cell cultures,

tissue engineering, medical and biological sensors, water

absorbent pads, hygiene products, breast implants, wound

A. Tripathi, A. KumarDepartment of Biological Sciences and Bioengineering, IndianInstitute of Technology Kanpur, 208016 Kanpur, IndiaFax: þ91-512-2594010; E-mail: [email protected]

Macromol. Biosci. 2011, 11, 22–35

� 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim wileyonline

dressing, and for enzyme and cell immobilization, etc.[1]

Although hydrogels have shown significant potential

towardsbiomedical andbiological applications, sometimes

conventional hydrogels do not meet the set criteria for a

specific application. For example, most hydrogels show

poor mechanical strength,[2] poor mass transport, and low

swelling behavior because of small pore architecture.[3] To

overcome these limitations, different kinds of techniques

have been applied tomodify thehydrogels, such as solvent-

casting particulate-leaching methods, gas foaming meth-

ods, fiber mesh approaches, freeze-drying methods, solu-

tion castingmethods, etc[3] and thesemethods enhance the

properties of hydrogels as a scaffold.

Recently, cryogel technology has emerged as a potential

approach to produce polymeric scaffolds, so called cryo-

gels.[4] The interconnected pore morphology in a cryogel

provides a capillarynetwork throughwhich the solvent can

library.com DOI: 10.1002/mabi.201000286

Multi-Featured Macroporous Agarose–Alginate Cryogel: Synthesis and Characterization . . .

www.mbs-journal.de

flow by convective mass transport. The macroporosity

allows unhindered influx of high-molecular-weight solutes

andnutrients, aswell as the transport of cellularwaste. This

also enhances the cellular proliferation and provides

increased matrix diffusion in the gel.[5] Classical hydrogels

swell slowly because of the low diffusion or low capillary-

driven absorption, whereas macroporous cryogels show

quick swelling kinetics with substantial viscoelastic

properties, which slightly vary with the material nature

and environmental conditions. With these key advantages

over hydrogels, cryogels have been used for various

applications in biotechnology and biomedical sciences.[6]

In particular, biomaterials based on natural polymers have

been frequently used for biological and biomedical

applications because of their good biocompatibility,

biodegradability, low toxicity, and other key advantages.

Ideally, materials should be designed and engineered in

such a manner so as to address the usage in more than one

particular application area. The selection of polymers for

scaffold synthesis is a crucial step while keeping in mind

these characteristics for providing an ideal scaffold/matrix

property.

Here we have selected two natural polysaccharides, i.e.,

agarose and alginate, for the synthesis of a proper matrix/

scaffold. Both are sufficiently biodegradable, biocompati-

ble, and inert bynature andhave soft tissue likemechanical

properties.[4] Because of these properties, agarose gels have

been investigated as carriers for various biomedical

applications.[7] On the other hand, alginate is regarded as

one of the most versatile polysaccharides considering its

large number of different applications.[8] On the basis of

these properties, these two polymers can have great

potential for bioengineering applications as a composite

scaffold. On the one hand, the homopolymer scaffolds of

agarosehave showna rigid gel structurewhile alginate gels

have a weak mechanical stability. However, the combina-

tion of the above properties of the polymers has not yet

been explored as a composite macroporous scaffold.

The goal of this study is to assess the macroporous

agarose–alginate (AA) matrix/scaffold for functional (bio-

logical, biochemical, and biomechanical) properties and its

multi-disciplinary applications in the bioengineering field.

The use of cryogel technology to produce a customized

novel AA scaffold with a controlled internal micro-

architecture, and characterized with respect to physical

and chemical means, will be addressed for various

applications.

Experimental Part

Materials

Low viscosity alginic acid sodium salt (from brown algae), N-(3-

dimethylaminopropyl)-N- ethylcarbodiimide hydrochloride (EDC;

www.MaterialsViews.com

Macromol. Biosci. 20

� 2011 WILEY-VCH Verlag Gmb

FW 191.71), Dulbecco’s modifiedmedium (DMEM), 4-6-diamidino-

2-phenylindole (DAPI), andpenicillin-streptomycinantibioticwere

purchased from Sigma Chemical Co. (St. Louis, MO, USA). Agarose

(low electroendoosmosis (EEO), gelling temperature �38–40 8C)was purchased from Sisco Research Laboratories (Mumbai, India).

N-Hydroxysuccinimide (NHS) was bought from Spectrochem

(Mumbai, India). Fetal bovine serum (FBS) was purchased from

Hyclone (UT, USA). Fibroblast (NIH-3T3) was procured from NCCS

(Pune, India). Other chemicals were of analytical grade and were

used without any further purification.

Synthesis of AA Cryogel Monolith

AA cryogels were synthesized using EDC with NHS for chemical

crosslinking. Low viscosity alginate solution (3.75%) was prepared

in a plastic tube (50mL) using deionizedwater as a solvent. On the

other hand, agarose (Low EEO; gelling temperature 38–40 8C)solutions (6% and 8%)were prepared in deionizedwater by putting

the agarose-containing plastic tube (50mL) in boiling water for

about 30min or until the solution become transparent. Four

milliliters of the stock solution of the alginate (3.75%) was then

added to 5mL of completely dissolved hot agarose solution (6% or

8%) and this wasmixed by vortexing. This heterogeneous solution

was incubated at 60 8C in a water bath for 5–10min to allow good

mixing of alginate with the agarose polymer chains. The mixture

was then taken out from the water bath and cooled at room

temperature. When the temperature of heterogeneous solution

reached 45 8C, 500mL of 0.35M freshly prepared EDC solution was

added, whichwas followed by adding 500mL of 0.2M NHS solution

and was thoroughly mixed by vortexing. The total volume of the

reaction mixture was 10ml, where the final concentration of

alginate was 1.5% and the final agarose concentration was either

3%or4%dependinguponthestockconcentrationsof6%or8%used,

respectively. The final crosslinker concentration of EDC and NHS

was 17.5�10�3 and 10�10�3M, respectively. The crosslinker-

containing heterogeneous reaction mixture of AA was added to a

5mLplastic syringeand immediately incubatedat–12 8Cfor16h ina liquid cryostat (Julabo, Seelbach, Germany). After incubation of

gels for overnight, they were then thawed in deionized water and

dried at room temperature for further characterization and use.

Synthesis of AA Cryogel Beads

The aqueous solution of alginatewasmixedwith a freshly prepare

aqueous solution of agarose (as mentioned above). The ratio of

agarose toalginateconcentrationwas4 :1.5and3 : 1.5. Themixture

of AA was incubated for 5 to 10min at 60 8C and then the

heterogeneoussolutionwas left to cool to roomtemperature.When

the temperature of the polymer solution came down to 45 8C, EDCfollowed by NHS was added andmixed by vortexing. The solution

was transferred into a disposable plastic syringe and added drop-

wise into moderately frozen paraffin oil. The added polymer

droplets took a round shape in paraffin oil. The whole solution

containing beads in paraffin oil was incubated at subzero

temperature, i.e., �20 8C for 16h and the beads were then taken

out from the oil. The washing of the cryogel beads was repeated

11, 11, 22–35

H & Co. KGaA, Weinheim23

24

www.mbs-journal.de

A. Tripathi, A. Kumar

using phosphate buffered saline (PBS, pH 7.4) overnight under

gentle magnetic stirring to remove paraffin oil completely.

Characterization of the Synthesized AA Cryogel and

Morphological Analysis

The characterization of AA cryogels was done using samples 5mm

in height and 13mm in diameter prepared from the monolith

cryogelandthemorphologicalanalysisofall thedesignedformatof

cryogels were also performed as per these dimensions.

SEM and Porosity Analysis

The morphology of the synthesized AA cryogels was analyzed by

scanning electron microscopy (SEM). SEM allows direct measure-

ment of porosity, average diameter of pores, and strut thickness by

image analyzing software. The cryogel sampleswere prepared and

vacuum dried overnight using vacuum desiccators to remove any

moisture content to make an effective coating for further high

resolution microscopy analysis. Samples were then coated with

gold using a sputter coater (Vacuum Tech, Bangalore, India). SEM

examinations were made on a FEI Quanta 200 at high vacuum at

20kV with spot size of 3.5mm.

The porosity of AA cryogels was theoretically calculated

according to Archimede’s principle. By this method, the dry mass

of cryogel was recorded. The test sample was submerged under

water in a specific gravity bottle and the submerged mass of the

cryogel sample was recorded. The cryogel was then taken out and

the wet mass was recorded. Triplicate samples were used for the

study. These values were used to calculate the porosity of the

cryogel by Equation (1):

Porosity ¼ Mw�Mdð Þ= Mw�Msubð Þ (1)

where, Mw is the water saturated wet mass of the cryogel, Md is

the drymass of the cryogel, and Msub is the submergedmass of the

cryogel.

Micro-Computed Tomography (m-CT)

The micro-computed tomography (m-CT) technique was used to

analyze the 3D architecture and structural homogeneity of the

porousmaterial.Analysisof theAAcryogelwas carriedoutusingm-

CT equipment (Skyscan 1174, Belgium) with associated software

NRECON. The m-CT technique provided important information

about the shape and compactness of material without physically

sectioning them.

Permeability and Flow Rate Measurement

The hydraulic permeability was determined using Darcy’s law to

describe the relationship of the liquid flow rate versus pressure in

polymericmonoliths. The cryogel samples (13mmdiameter, 5mm

height) were prepared and placed in the permeability measure-

ment setup.[9] Under a constantwater pressureheadappliedon the

Macromol. Biosci. 2


porous material in the experimental setup, the water flushed out

from the outlet in 2min was collected and weighed. While in a

control experiment, a cryogelwas not put in the experiment setup.

The permeability was calculated by applying the recorded data to

Equation (2):[9]

011, 11,

H & Co

k ¼ DX

A � MB2� 2p2r4

MB1=MB2ð Þ2�1(2)

Where, K is the hydraulic permeability of the porous material, A is

the flushing area of the cryogel, DX is the thickness of the scaffold,

MB1 is the mass flow rate from the outlet of the control setup, and

MB2 is the mass flow rate from the outlet of the test setup.

The flow rate measurement was done using a peristaltic pump,

which was calibrated accordingly to Adrados et al.[10] against the

flow ratewithout connecting the test sample in between the path.

Thewaterflowrateof theAAcryogelwasdeterminedby taking the

same size (1.3 cm diameter, 2 cm height) cylindrical monolithic

cryogel in a swollen state (water saturated) and inserting it into the

same size of plastic syringe. The inlet and outlet of the syringewas

connected to the preset pumpand registering themaximumwater

flow limit (mL �min�1) of samples till the test sample was not

showing any back pressure.

Swelling Kinetics and Swelling Ratio

The swelling kinetics was carried out using a conventional

gravimetric procedure.[11] This method was used to determine

thewateruptake capacity of aporousmaterialwith respect to time

at a particular temperature. The equilibrium swelling measure-

ments were carried out in deionized water at room temperature

(�25 8C). Cryogel sections of 13mm diameter and 5mm height

were prepared from a cylindrical monolith cryogel. All sections

were dried at 60 8C in an oven. The initial dry weight of all the

cryogel sections was measured and then samples were placed in

deionized water. The samples were removed from the water and

weighed at regular time intervals. At least four samples with a

similar height and diameter were used for the study. The

percentage water uptake capacity was determined using

Equation (3):

Wu ¼ 100� Wt�Wg

� ��We

� �(3)

where Wu is thewater uptake capacity, Wt is the weight at regular

time intervals, Wg is the weight of the dry cryogel, and We is the

weight of water in the swollen gel at swelling equilibrium at a

particular temperature.

However, the swelling ratio (SR) is an important parameter

which reveals the solvent absorption capacity of sample. It is

calculated as per Equation (4):

SR¼ Ws�Wdð Þ=Wd (4)

where Ws is the weight of the swollen gel and Wd is the weight of

the dry gel.

22–35

. KGaA, Weinheim www.MaterialsViews.com


www.mbs-journal.de

Density Measurement

The ratio of the wet weight as well as dry weight of the cryogels

with respect to their volume was used to obtain the wet and dry

densities of the test samples. The apparent density in g � cm�3 was

calculated from Equation (5):

www.M

r ¼ W=p� D=2ð Þ2 � H (5)

ere, r is the apparent density, W is the weight of the cryogel
Wh
sample in grams,D is the diameter of the sample in cm, andH is the

thickness of the sample in cm.

Mechanical Analysis

Unconfined Compression Analysis

For the initialmechanical characterization of the cryogels, uniform

gel samples were prepared. The height and diameter of the disc-

shaped samples were 5mm and 13mm, respectively. Cryogel

samples were saturated with PBS (pH 7.4) and were tested in

unconfined compression using a custom computer controlled

mechanical testing system (Zwick/Roell Z010, Germany). The

sampleswereuniaxially compressedbyplacingbetween twoarms

of a load frame. The test were conductedwith a 10 kN load cell at a

ramp rate of 1mmmin�1 up to 90% strain of the total length of the

samples at room temperature (�25 8C). At least four samples were

used to calculate the average unconfined compressive modulus

from the stress (kPa) and strain (%) graph shape of unconfined

mechanical test.

Cyclic Deformation Analysis (Fatigue Test)

AA cryogel samples weremade according to themethod described

earlier. The test samples of similar size (13mmdiameter and 5mm

height) were prepared from the freshly synthesized monolith.

These cryogel sampleswereaxially compressedup to40%strain for

100 000 cycles in deionized water at room temperature (�25 8C).Cyclic deformationwasmeasured for each sample across the range

of2and5HzonMTS,810MaterialTestSystem(USA).Aplasticplate

surface with circular grooves filled with water was used on the

testing apparatus to keep the samples stationary and saturated

during testing. The mass and dimensions of the samples were

measuredbeforeandafter fatiguecycling todetermine theeffect of

cyclic deformation.

In vitro Degradation Measurement

Thescaffoldwaspreparedasa5mmdisk format,withadiameterof

13mm from the cylindrical monolithic cryogel and then sterilized

by1h incubation in70%ethanol. The sampleswereplaced in50mL

tightlycappedplastic tubes,eachtubecontaining10mLof0.1MPBS

(pH 7.4). The samples were incubated at 37 8C for various time

periods up to eight weeks. At each time point, two samples were

taken out from the PBS, washed with distilled water, and air dried

overnight at room temperature. The corresponding initial dry

weight (WI) andfinal dryweight (WF)was recordedbeforeandafter

incubationof the samples and theweight losswas calculatedusing

aterialsViews.com



Equation (6):

11, 11,

H & Co

Weight loss %ð Þ ¼ WI�WFð Þ=WI½ ��100 (6)

Microbial Cell Immobilization

For cell adherence studies, AA cryogel discswere prepared from the

cryogel monolith. The discs were sterilized by incubating in 70%

ethanol for 2 h. After incubation, samples were kept in vacuum

desiccators overnight to remove ethanol content completely. On

the other hand, E.coli cell (strain BL-21) culture in LBmedium in the

presence of ampicillin was setup. The bacterial culture was

incubated at 37 8C till its optical density reached 0.4 at 600nm.

Each cryogel disc pre-saturated in sterile PBS was incubated with

one milliliter of active bacterial culture broth for 15 to 20min at

room temperature inside the laminar hood. The sampleswere then

removed and gently washed with sterile PBS for 5min. For

analyzing the microbial cell adherence, the cryogel sections were

immediately fixed using 2.5% glutaraldehyde solution for over-

night incubation at room temperature. After incubation, sections

were again gently washed with sterile PBS (pH 7.4) followed by

dehydration by gradually increasing the concentration of ethanol

from20 to 100%with10% increments and the incubation timewas

10min in eachdehydration step. The sectionswere thenallowed to

dry at room temperature to evaporate ethanol content from the

samples. For entrapment, the bacterial cell mass (25mg) was

dissolved in 10mL of polymer mixture before and after bead

preparation and to fix the cells asmentioned earlier. The surface of

dried cryogel sections was analyzed by SEM after gold coating.

Mammalian Cell Culture

The cryogel sections were cut into two different thicknesses. The

5mm height and 13mm diameter sections were used for cell

viability/proliferation analysis, while thin 100mm sections were

used for fluorescent microscope observations. The thin sections

wereused for thefluorescent studybecause the ease of penetration

of light facilitates better imaging and understanding of the

growing cells. The sampleswere sterilizedwith70%ethanol for 4 h.

Samples were taken out from ethanol and kept in a 24-well tissue

culture plate inside a laminar hood. All the samples were gently

washed four times with sterile PBS (pH 7.2). Each step of washing

was for 10–15min. Before seeding, sampleswere equilibratedwith

Dulbecco’s modified eagle’s medium (DMEM) that contained 10%

fetal bovine serum and 1% streptomycin/penicillin for 4h.

Fibroblasts (NIH-3T3) were cultured and cell growth was

observed under a fluorescent microscope using 100mm sections,

while 5mm thick samples were used for cell proliferation assays.

Fibroblast cells were suspended in culture medium and were

seeded drop-wise to each AA cryogel sample (50mL on 100mm

sections and 500mL on 5mm thick sections; cell density 1�106 cellsmL�1). The cell-seeded scaffold sections were placed in a

humidified 5% CO2 atmosphere at 37 8C for up to one week. The

culture medium was exchanged with the fresh medium every

alternate day during the cell culture period.

22–35

. KGaA, Weinheim25

26

www.mbs-journal.de


Cell Viability/Proliferation Analysis (MTT Assay)

The effect of the hybrid AA cryogel on themetabolic activity of the

fibroblasts (NIH-3T3)wasaccessed invitro at apredetermined time

intervals, i.e., every alternate day up to one week. The cellular

activitywasmeasured at a physiological pHof 7.4using a standard

protocol of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium

bromide (MTT) assay to determine the optical density of the end

product at 570nm.[4]

Heavy Metal Binding

In the initial experiment, the potentiality of the AA cryogel was

examined for heavy metal binding and recovery by visual

observations. Copper sulfate and nickel sulfate solution (0.5M)

were used in these experiments. The AA cryogel monoliths (1.3 cm

diameter and 3 cm height) were incubated in both copper sulfate

andnickel sulfate solution for 1 h each. The cryogelmonolithswere

taken out in fresh tubes and washed with PBS to remove the non-

specificallyboundmetal solution.The repeatedlybindingofmetals

wasevaluatedby incubating themetalboundmonolith in0.1MHCl

solution to recover the bound metal and again incubated in the

same metal solutions for up to five cycles.

Results and Discussion

Synthesis of AA Cryogels

AAcryogelswere synthesized by varying the concentration

of agarose in the co-polymer and using crosslinking agents

like EDC andNHS. The synthesis is based on cryogelation of

agarose and alginate chains at subzero temperatures in the

presence of a crosslinker. The synthesized AA cryogels are

white in color and retained a three-dimensional architec-

ture even on air drying (Figure 1). On complete drying these

cryogels has shrunk approximately 1mm in size from the

original size of 13mm diameter and 5mm height. While

soaking in water they return into their original shape and

size. The crosslinking reaction between agarose and

alginatepolymer chainsusingEDChasnotbeen specifically

studied yet, but previous studies suggest that EDC may be

involved in the crosslinking within the carboxy group rich

Figure 1. The digital images of different format of AA cryogelshowing a) sheet, b) disk, c) monoliths, and d) beads.

Macromol. Biosci. 2


in alginate chains,[12] or in acid anhydride formation

between two carboxy groups of alginate by the action of

EDC and eventually the resultant acid anhydride may

readily react with a hydroxy group of agarose to form an

ester bond.[13] The use of NHS to improve the performance

of EDC crosslinking iswell documented in the literature.[14]

Therefore, the reaction conditions for the crosslinking of

agarose and alginate with respect to crosslinker concentra-

tionswereoptimizedbyphysicalobservationsby thenaked

eyeof thesynthesizedmatrices inorder toachieveascaffold

with good mechanical integrity. In addition, initial experi-

ments with different concentrations of agarose and

alginate polymers were attempted to synthesize the best

mechanically stable cryogel with high macroporous

architecture. We selected 3:1.5% and 4:1.5% agarose to

alginate ratios in the cryogels, respectively, for further

study, because they showed good stability and a better

macroporous structure than the other compositions (data

not shown). The characterizations of the matrix and its

screening for various applications were performed using

these two compositions.

In a separate set of control experiments in order to check

the crosslinker effect, agarose and alginate aqueous

solutions were mixed a) without using crosslinker, b) only

in thepresenceofEDC,andc)only inthepresenceofNHS.All

the combinations of polymer mixture were incubated in a

5mL plastic syringe at –12 8C for 16h. After completing the

appropriate time of incubation period, gels were thawed in

deionized water at room temperature. In all the sets of

experiments including thecontrol, itwasobserved thatgels

could be formed but did not have the properties of the

cryogels of AA synthesized in the presence of EDC and NHS

together. All the control gelswere brittle in naturewith low

elasticity andhad small pores likeahydrogel synthesizedat

room temperature, while the AA cryogels synthesized at

subzero temperatures in the presence of EDC andNHSwere

soft, spongy, elastic and had a macroporous architecture.

This particular polymer combination was used to synthe-

size cryogels in other types of formats, i.e., monoliths, disks,

sheets, and beads (Figure 1). These formats can have

significant potential for various biological applications.

Monoliths and beads can be used for direct recovery of a

product from an immobilized cell system in a continuous

system, while the disk and sheet formats of the gels can be

used as cell supports in three-dimensional architecture for

tissue engineering applications. Agarose itself can by

physically crosslinked and make a porous three-dimen-

sional scaffold while the presence of alginate in the

composite increased the softness of the cryogel as well as

the spongynature.On theotherhand, thealginatepresence

is also beneficial to cultivate different cell types for tissue

engineering applications[15] and immobilization pur-

poses.[16] Alginate has also been used to capture heavy

metals and bacteria from waste water.[17]

011, 11, 22–35

H & Co. KGaA, Weinheim www.MaterialsViews.com

Figure 2. SEM image of AA cryogel. a) AA cryogel containing 3%agarose (ratio; 3 : 1.5%) and b) AA cryogel containing 4% agarose(ratio; 4 : 1.5%). Both (a) and (b) show low magnification images intheir insets, suggesting a uniform pore distribution within themonoliths.


www.mbs-journal.de

Morphological Analysis (SEM, Porosity, and m-CTAnalysis)

Microstructure analysis of macroporous AA cryogels was

evaluated by various microscopic observations. The SEM

observations of various formats of the AA cryogel showed

slight variation in their microstructure (Figure 2). The

monolith sections of the cryogel showed a comparatively

smaller pore diameter than thebead format. Thepore range

of cryogel sections of the monolith was 40 to 200mmwith

an average pore diameter in the approximate range of 80 to

125mm in 3:1.5% AA. On the other hand, AA (4:1.5%)

showed a pore size of 30 to 160mm and an average pore

diameter lying in the range of 60 to 85mm. The pore size

range of the cryogel sheet was more or less in the same

range as that of the monolithic cryogels. The pore range of

the internal section of the cryogel beads was 60 to 180mm




and the average pore diameter was lying in the range of 95

to 127mm.

TheSEMobservationshowedauniformporedistribution

in the whole monolithic scaffold of both compositions, i.e.,

3:1.5% and 4:1.5% (Figure 2a and 2b). The pore diameter

range was higher in the 3:1.5% monolith in comparison to

4:1.5%. This might be because of the variation in agarose

polymer concentration, whereas, the formation of bigger

pores in beads compared to themonolith and sheetmay be

because of the better heat transfer abilitywithin the beads.

The size of the beads and the method used for bead

formation provide a high surface area and surroundings

within the cryogelation system, which can better circulate

and maintain the temperature. On the other hand, during

the synthesis of the monolith and sheet, the moulds used

can slightly affect theproper transport of temperature from

the cooling system to polymer system. In the cryogel

formation, a proper cooling systemhas an important role in

the formation of the desired cryogel. The rate of ice crystal

formation and crosslinking/gelation of the polymer or

polymer mixture can occur simultaneously. At subzero

temperatures most of the solvent is frozen, while that part

of solvent left unfrozen is called the unfrozen liquid

microphase. The dissolved substances in the unfrozen

phase concentrate and undergo chemical reactions, which

leads to gel formation. The ice crystals of solvent in the

frozenphase act as porogens. After thawing the ice crystals,

a system of a large interconnected porous network is

formed within the gel. So, during the cryogelation process,

variations in the temperature and other parameters can

change the structural homogeneity of a cryogel. In Figure 3,

micrographs of cryogel beads revealed that the outer

surfacewasquite different than the internalmorphologyof

the surface. The outer surface of the bead has smooth nano-

grooves (Figure 3a) although the inner part has a macro-

porous architecture (Figure 3b). This morphology may

provide a novel system to immobilize the cells or enzyme

for effective recoveryof theproduct. Thegroovespresent on

the surface of the cryogel beads help the diffusion of

product from inside the bead to the medium outside

without cell leakage. On the other hand, variation in the

agarose ratio during the cryogel synthesis has shown a

change in the property of the cryogel structure. The SEM

observation of theAAcryogel in the sheet format suggested

that it has a uniform porous architecture (Figure 3c) and

these pores were interconnected (Figure 3d, where (d) is a

magnified image of (c)). During the observations, cryogel

test samples that have a higher agarose content (4%)

showed a tight and smooth porous architecture than low

agarose-containing cryogels, i.e., 3%. The higher concentra-

tion of agarose in the composite also increased the wall

thickness as well as mechanical stability of the gel. The

cryogels show good interconnectivity revealed by inverted

microscope observations using ethidium bromide (EtBr)

11, 11, 22–35


Figure 3. SEM images of cryogel beads and sheet. a) The outer part of the cryogel bead and b) the inner surface with correspond inset imagesat high magnification. The inset image of (a) shows nanometer-range grooves on the outer surface and the inset image of (b) suggeststhe internal macroporous bead structure. c) The side view micrograph of the sheet format of AA cryogel sheets containing 3% agarose.d) A magnified image of (c) with uniform pore distribution.

28

www.mbs-journal.de


staining of 100mm sections of the 4:1.5% cryogel (Figure 4).

Porosity of the synthesized cryogels was measured using

Archimedes’s principle. Both types of cryogel monoliths

showed good porosity which was 91.47% and 84.69% in

3:1.5% and 4:1.5% cryogels, respectively (Table 1).

In the present study,m-CT is originally used as an

additional tool to accompany the SEM structural delinea-

tion (Figure 5). The three-dimensional plot of a monolith

(Figure 5d1) and sheet (Figure 5d2) form of AA cryogels

presented a percentage volume of a scaffold to determine

the structural homogeneity and also the difference in their

morphology even after a change in the synthesis format.

The micro-computed based observations showed that the

mean porosity of the cryogelmonoliths and sheets (4:1.5%;

agarose/alginate) was more than 85% and the mean pore

size was up to 122.6mm. The swelling degree of the cryogel

does notmake anynoticeable difference in themorphology

observed by the 3D plotted construct of scaffolds. The 2D

Macromol. Biosci. 2


histomorphometric analysis allowed for comparing the

structural integrity of the cryogel (Figure 5b and 5c).

Nevertheless, these morphological studies compliment

these novel polymeric scaffolds for fostering various

bioengineering applications, where the macroporosity is

a very important parameter, such as for tissue engineering,

bioreactors, bio-separation applications, etc.

Permeability and Flow Rate Measurement

Previous studies on scaffold design and characterization

have revealed that besides porosity, other parameters such

aspore interconnectivityandpermeabilityaffectmolecular

transport.[4,5] The highly porous scaffold should also have

interconnected pores, thus increasing the diffusion effi-

ciency as well as permeability. The calculated average

hydraulic permeability of the monolith of AA cryogels

(ratio; 3:1.5% and 4:1.5%) was 1.1� 10�9 and

011, 11, 22–35


Figure 4. Inverted microscope image of thin cryogel sections; a) unstained 20mm section and b) EtBr stained 100mm section, showingoverall interconnectivity within the gel.

Table 1. Characteristics of AA cryogels prepared in different concentrations.

Conc.

of AA

cryogel

Average

porosity

Average

swelling

ratio

Density Permeability Flow

rate

Average

degree of

degradation

% g � cm�3 m4 �N�1 � s�1 mL �min–1 %

Dry Wet

3 : 1.5% 91.47 21.21� 0.23 0.029� 0.001 0.887� 0.039 1.1� 10�9 Up to 10 39.68� 1.68

4 : 1.5% 84.69 17.02� 0.32 0.042� 0.007 0.949� 0.020 7.6� 10�10 Up to 10 41.00� 1.03


www.mbs-journal.de

7.6� 10�10m4N�1 � s�1, respectively. It can be seen that the

hydraulic permeability of the scaffolds is very efficient,

which can provide good porosity during bio-transport

processes to produce neo-tissue, or in continuous bioreac-

tors for the production and recovery of active biomolecules.

The flow rate of the AA cryogels was measured by flow

rates of water across the monolithic cryogels using a

peristaltic pump. The AA cryogels have shown good

convective flow of water as the flow rates went up to

10 mLmin�1 (Table 1). It is desirable that the convective

flow rate of the scaffold should be known, which will be

useful for cell seeding in a perfusion bioreactor or for tissue

engineering applications. This underpins the understand-

ing of the biotransport processes in the scaffolds during

tissue engineering applications. This can also be important

because an excessively high seeding density or cell growth

increases the risk of cell death if the scaffold shows high

resistance against the flow of medium.

Swelling Kinetics and Swelling Ratio

Swelling characteristics are important parameters that

influence the biological functionality of a scaffold. The




swelling kinetics of AA cryogels was studied by conven-

tional gravimetric method. The AA cryogels synthesized in

different formats did not show any significant differences

in their swelling kinetics as shown in Figure 6. The rate of

water uptake capacity was approximately the same in the

both types of cryogels (3:1.4% and 4:1.5%). These cryogels

swelled up to 92–95% of their capacity within 30 s at room

temperature (�25 8C). However, both the cryogel systems

attain equilibrium within 1min. Apart from that, the

swelling ratio of different ratios of cryogels showed some

minor differences which are shown in Table 1, where the

cryogels with 3:1.5% and 4:1.5% concentration showed

average swelling ratios of 21.21� 0.23 and 17.02� 0.32,

respectively.

Density Analysis

The density of the dried as well as the wet AA cryogel

samples were calculated by measuring their weight and

dimensions, which are shown in Table 1. The AA cryogels

showed a density less than the apparent density of water,

i.e., 1 g cm�3 at room temperature, and because of this AA

cryogels float in water. It was also noticed that on

11, 11, 22–35


Figure 5. Micro X-ray computer tomographic scan of an AA cryogel. Scaffold discs were scanned at 11.5mm resolution. a) Transmissionimages, b) a reconstructed image, c) a binarised image, and d) three-dimensional (3-D) images (d1; monolith and d2; sheet) of an AA cryogel.The white area on the images shows the scaffold material and the black areas refer to the void space. In (a), arrow 2 shows the tack ontowhich the scaffold (arrow 1) was attached for scanning. Arrow 3 shows the sample holder. Scale bars are equal to 800mm.

Figure 6. Swelling kinetics of the AA cryogel showing a very highwater uptake capacity (Wu %), where both ratios, i.e., 3 : 1.5% (-&-)and 4 : 1.5% (-^-) show the same swelling rate and wereequilibrated within 1 min.

30

www.mbs-journal.de


increasing the concentration of polymer the density of the

sample also increased correspondingly because density is

dependent upon mass and volume. But above an optimal

concentration of polymers, it was observed that cryogela-

tion could not occur uniformly within the gel at subzero

temperature. However, these cryogels show a high density

Macromol. Biosci. 2


with low porous architecture and caused immersion in

water.

Unconfined Compression Analysis

Mechanical characterizationunder unconfined compression

of theAAcryogels typically shows anon-linear stress–strain

response in a distinct upward trend (Figure 7). The

observation of dynamic strength at the strain value up to

90%showednodestruction in themorphologyof the cryogel

sections. The secant compressivemoduluswas calculated as

stress/strain at 15% compression of the total length of the

test sample. The compressive modulus of the AA cryogel of

both combinations, i.e., 3:1.5% and 4:1.5% was 67.73� 1.36

and 73.34� 2.02 kPa, respectively. The compressive moduli

of the cryogel samples containing 4% agarose showed a

higher strength than the gel which contains a low agarose

concentration. Previousstudies suggested that on increasing

the polymer concentration during scaffold synthesis it

proportionally increases its stiffness but also increases the

brittlenessdependinguponthenatureof thepolymersbeing

used. To recognize the importance of mechanical stimuli in

tissue engineering and stress reactions in a bioreactor, it

becomes important to develop a scaffold material that

maintains itsmechanical integrity duringmechanical strain

applications in the bioengineering arena. Here the AA

011, 11, 22–35


0 20 40 60 80 1000

20

40

60

80

Stre

ss (k

Pa)

Strain (%)

Figure 7. Unconfined compressive stress–strain graph showing anon-linear upward increment of the cryogel showing mechanicalstrength up to 70% compression of their original length. There-after, the stress was transferred from the cryogel sample to theinstrument fixture which could be seen from the steep increase instress. Where, 3:1.5% (-*-) and 4:1.5% (-&-).


www.mbs-journal.de

cryogels showed a substantial mechanical property to

immobilize/cultivate the cells in 3D environments. This

can be followed by transferring to patient to regenerate the

neo-tissue or recover the active biomolecules from the

bioreactor system.

Cyclic Deformation Analysis (Fatigue Test)

In tissue engineering, cyclicmechanical strain–restrain has

been shown to be important to deliver mechanical signals

duringneo-tissuedevelopment.[18] Severalpreviousstudies

have shown the importance of cyclic stress–strain in the

improvement of a tissue engineered construct either by

influencing the mechanical strength of the construct or by

Figure 8. The digital images showing the initial shape of an AA cryogel disc and acomparison of images after two months of in vitro incubation in PBS at 37 8C.

stimulating the production of extracel-

lular matrix (ECM) during in vitro cul-

ture.[19] The PBS-saturated (0.1 M, pH 7.4)

AA cryogels were compressed at the

frequencies of 2 and 5Hz with varying

strains (10%, 20%, and 40%) up to 1� 105

cycles to check for any permanent

deformation and destruction at these

conditions within the gel. The post-

fatigue observations revealed that there

were no noticeable changes found in the

weight and dimensions of the test

samples compared with the initial read-

ings. In contrast, at high frequency (5Hz)

and high strain (40%) some minor cracks

were found on the peripheral surface of

the test samples but no permanent




deformations ormajor crackswere observed. Alginate does

not exhibit a pronounced fracture property because of its

well knownelastic property,while by combining agarose it

provides stiffness to the hybrid cryogel and thus suitable

mechanical stability. The stability of AA cryogels as

described here suggests its potentiality for tissue engineer-

ing applications.

In vitro Degradation Measurement

The degradation kinetics of AA cryogelswas observed up to

eight weeks of incubation of test samples at 37 8C in PBS

(0.1 M; pH 7.4) in sterile conditions. The degradation rate

wascalculatedbyachange indryweightof the test samples

at each pre-defined time interval (Figure 8). The degree of

degradation of the AA cryogels, 3:1.5% and 4:1.5%, was

39.68� 1.68% and 41� 1.03%, respectively (Table 1). These

gels showed good degradation rate kinetics as shown in

Figure 9. As a hydrophilic polymer in a hybrid gel, it is

presumed that the macromolecules (long polymer chains)

of the cryogel surface undergo preferential hydrolytic

scission into smaller molecules (oligomeric units), which

can diffuse out into the PBS. The rate of degradation was

optimum as it was not too quick and not too slow. So, this

type of property of the hybrid scaffold demonstrates its

potentiality for other bioengineering applications.

Analysis of Microbial Cell Immobilization

During the past two decades a significant amount of

research has been performed on the use of alternative

fermentation and product recovery techniques in the

industrial biotechnology processes using microorganisms.

Such classical fermentations suffer fromvarious constrains

such as low cell density, nutritional limitations, and batch-

mode operationswith high down times.[20] The continuous

fermentationswith free-cells andcell recycleoptionsaimto

11, 11, 22–35


Figure 9. The degree of degradation of an AA cryogel (both ratio)showing approximate uniform degradation kinetics. Where (-&-)is a cryogel containing 4% agarose and (-^-) is a cryogel contain-ing 3% agarose.

32

www.mbs-journal.de


enhance the cell population inside the fermentor. Immo-

bilization systems have examined the use of immobilized

cells by twomain techniques, adsorption and entrapment.

Our initial experiment showed that E.coli.-BL21 cells could

nicely adhere and were entrapped within the scaffold

surface (Figure10). Thisapproachwasaverysimplemethod

with highmass transport and cell interaction ability. These

studieshaverevealedpossibleareas for the improvementof

productivity and its recovery.

Mammalian Cell Culture

Fibroblast (NIH-3T3)-seeded cryogel samples were exam-

ined up to the 7th day of culture. Examination revealed that

NIH-3T3 could nicely adhere and proliferate on 100mm

Figure 10. SEM micrograph of microbial cell immobilization on the Aculture. a) The control, b) cell adhering on the whole surface of the ge21) adherence at high magnification (4 000�).

Macromol. Biosci. 2


cryogel sections (Figure 11). The cell proliferation was

examined by nuclear staining using 200 ngmL�1 working

solution of 40,6-diamidino-2-phenylindole (DAPI) prepared

in PBS. Cell-cultured cryogel sections were incubated with

DAPI solution for 5min and then gently washed with PBS.

The cryogel sections without cells were also stained with

DAPI,whichdidnot showanyfluorescence (Figure11a). The

morphology of the cell nuclei was observed after 24h and

on the 7th day of cell culture using a fluorescence

microscope at an excitation wavelength of 350nm. After

24h, cells could adhere nicely to the scaffold surface

(Figure 11b) and increased their cell number on the 7th day

of cell culture (Figure 11c).

Cell Viability/Proliferation Analysis (MTT Assay)

The AA cryogels showed an increasing cellular metabolic

activity with time, as shown in Figure 12, while the control

(2D; 24-well tissue culture plate) wells showed increasing

cellular activity up to the 3rd day of cell culture and after

that the cell viability started decreasing and declined

drastically at the 7th day of cell culture. It might be because

the growing cells reached their confluency in the control

wellswithin5days andafter that they compete for survival

and start dying, which may cause the reduction of

metabolic activity in the 2D control wells. In contrast,

cryogel samplesprovidedahighenoughsurfacearea for cell

proliferation. The effective growth of fibroblasts on the AA

cryogel showed the cellular compatibility of these scaffold

for neo-tissue development.

Heavy Metal Binding

Extensive research is being focused on the removal and

recovery of heavymetals fromwastewater.[17] In the initial

experiment, binding of heavy metals on a AA cryogel

monolithwasexaminedusingtwosolutions, i.e., CuSO4and

A cryogel. The cryogel samples were treated with active bacteriall at low magnification, and c) confirmation of bacterial cell (strain-BL

011, 11, 22–35


Figure 11. Fluorescent microscope images of fibroblast (NIH-3T3)seeded on a 100mm thin cryogel section analysed by nuclearstaining using DAPI stain. a) Background staining of the cryogelscaffold without cells, b) cell adherence after 24 h of seeding, andc) high cell growth covering the whole scaffold after 7 d of cellculture.

Figure 12. The relative viability of fibroblasts (NIH-3T3) as deter-mined by MTT assay. Cells were grown up to one week in apolystyrene-coated tissue culture plate well which is used as acontrol (dotted bar), a cryogel containing 3% agarose (stripedbar), and a cryogel containing 4% agarose (blocked bar). Theabsorbance of blue formazan was measured at 570 nm at differ-ent time intervals up to one week culture.


www.mbs-journal.de

NiSO4 (Figure 13). Visual observation showed that the color

of the cylindricalmonoliths of theAAcryogel changed from

white (Figure 13a and 13c2) to blue when treated with

CuSO4 (Figure 13c1) and green when treated with NiSO4

(Figure 13c3). This indicated the affinity binding of the

heavy metals on the cryogel monolithic column. The

system is more beneficial as it can be reused for repeated




metal binding. For checking its reusable potentiality, we

tried repeated binding of CuSO4 up to five times. A HCl

solution (0.1 M) was used for the recovery of copper. The

appreciable binding of copper in each cycle was visually

observed. However, further optimizations are required to

increase and quantify the metal binding and recovery

efficiency of the gel.

Conclusion

In this study a novel hybrid AA cryogel is successfully

synthesized. The biocompatible and biodegradable AA

scaffold showed unique properties for bioengineering

applications. The study suggested that these cryogels

showed a macroporous interconnected architecture, high

swelling kinetics, and amiable mechanical properties

proving its stability and application in tissue engineering

for in vitro neo-tissue development. Experimental results

also showed that theAAcryogel canhavea role as a support

matrix for cell immobilizationand for theeffective recovery

of a product from a medium, and also as a filter to remove

heavy metals from wastewater with reusable properties.

The goal of the study was to design such a multi-featured

matrix for the various applications that have been explored

here. The materials were characterized thoroughly for

desired applications. However, further optimizations may

be required for a particular area of interest to have an in-

depth evaluation.

11, 11, 22–35


Figure 13. Digital photographs of dry monoliths of an AA gel; a) synthesized at subzero temperature, and b) synthesized at roomtemperature. These gels were incubated with copper sulfate and nickel sulfate solutions and both the copper (c1) and nickel (c3) bindingability is shown along with its control (c2).

34

www.mbs-journal.de


Acknowledgements: The authors acknowledge the financialsupport received from Department of Biotechnology (DBT),Department of Science and Technology (DST), Ministry of Scienceand Technology, Government of India, and Protista BiotechnologyAB, Lund, Sweden. A Research Fellowship to A.T. from the Councilfor Scientific and Industrial Research (CSIR), India, for DoctoralResearch work is duly acknowledged.

Received: July 9, 2010; Revised: August 20, 2010; Publishedonline: November 15, 2010; DOI: 10.1002/mabi.201000286

Keywords: agarose-alginate blend scaffolds; biomaterials; cryo-gels; heavy metal binding; immobilization; tissue engineering

[1] K. Park, Drug Delivery Technol. 2002, 2, 38.[2] H. T. Peng, L. Martineau, P. N. Shek, J. Mater. Sci. Mater. Med.

2007, 18, 975.[3] E. Sachlos, J. T. Czernuszka, Eur. Cell Mater. 2003, 5, 29.[4] A. Tripathi, N. Kathuria, A. Kumar, J. Biomed. Mater. Res.

Part A 2009, 90, 680.[5] N. Kathuria, A. Tripathi, K. K. Kar, A. Kumar, Acta Biomater.

2009, 5, 406.[6] [6a] V. Lozinsky, I. Y. Galaev, F. M. Plieva, I. N. Savina,

H. Jungvid, B. Mattiasson, Trends Biotechnol. 2003, 21, 445;[6b] S. Nilsang, K. S. Nandakumar, I. Y. Galaev, S. K. Rakshit,R. Holmdahl, B. Mattiasson, A. Kumar, Biotechnol. Prog. 2008,23, 932; [6c] P. Arvidsson, F. M. Plieva, I. N. Savina, V. I.Lozinsky, S. Fexby, L. Bulow, I. Y. Galaev, B. Mattiasson,J. Chromatogr. A 2002, 977, 27; [6d] A. Kumar, P. Fatima,I. Y. Galaev, B. Mattiasson, J. Immunol. Methods 2003, 283,185; [6e] V. I. Lozinsky, F. M. Plieva, Enzyme Microb. Technol.1998, 23, 227; [6f] L. Doretti, D. Ferrara, P. Gattolin, S. Lora,F. Schiavon, F. M. Veronese, Talanta 1998, 45, 891; [6g]K. Bloch, V. I. Lozinsky, I. Y. Galaev, K. Yavriyanz,M. Vorobeychik, D. Azarov, L. G. Damshkaln, B. Mattiasson,P. Vardi, J. Biomed. Mater. Res. Part A 2005, 75, 802; [6h]

Macromol. Biosci. 2


M. Dainiak, A. Kumar, I. Y. Galaev, B. Mattiasson, Proc. Natl.Acad. Sci. USA 2006, 103, 849.

[7] [7a] J. Liu, L. Li, Eur. J. Pharm. Sci. 2005, 25, 237; [7b] A. Jain, Y. T.Kim, R. J. McKeon, R. V. Bellamkonda, Biomaterials 2006, 27,497; [7c] H. Yang, H. Iwata, H. Shimizu, T. Takagi, T. Tsiji, F. Ito,Biomaterials 1994, 15, 113; [7d] S. Sakai, K. Kawabata, T. Ono,H. Ijima, K. Kawakami, Biomaterials 2005, 26, 4786; [7e]H. Gruber, G. L. Hoelscher, K. Leslie, J. A. Ingram, E. N. Hanley,Biomaterials 2006, 27, 371; [7f] H. E. Gruber, E. C. Fisher,B. Desai, A. A. Stasky, G. L. Hoelscher, E. N. Hanley, Exp. Cell.Res. 1997, 235, 13; [7g] W. Gu, H. Yao, C. Y. Huang, H. S.Cheung, J. Biomech. 2003, 36, 593; [7h] R. Mauck, M. A. Soltz,C. C. B. Wang, D. D. Wong, P.-H. G. Chao, W. B. Valhmu, C. T.Hung, G. A. Ateshian, J. Biomech. Eng. 2000, 122, 252.

[8] [8a] K. I. Draget, O. Smidsrod, G. Skjak-Braek, Alginates fromAlgae, 1st edition, Wiley-VCH, Weinheim 2005, p. 1; [8b] D. A.Lee, T. Reisler, D. L. Bader, Acta Orthop. Scand. 2003, 74, 6.

[9] J. Li, A. F. T. Mak, J. Biomater. Appl. 2005, 19, 253.[10] B. Adrados, I. Y. Galaev, K. Nilsson, B. Mattiasson,

J. Chromatogr. A 2001, 930, 73.[11] A. Srivastava, E. Jain, A. Kumar, Mater. Sci. Eng. A 2007, 464,

93.[12] S. N. Park, H. J. Lee, K. H. Lee, H. Suh, Biomaterials 2003, 24,

1631.[13] [13a] S. N. Park, J. C. Park, H. O. Kim, M. J. Song, H. Suh,

Biomaterials 2002, 24, 1205; [13b] Y. S. Choi, S. R. Hong, Y. M.Lee, K. W. Song, M. H. Park, Y. S. Nam, Biomaterials 1999, 20,409.

[14] [14a] J. M. Lee, H. H. L. Edwards, C. A. Pereira, S. I. Samii,J. Mater. Sci. Mater. Med. 1996, 7, 531; [14b] D. Sehgal, I. K.Vijay, Anal. Biochem. 1994, 218, 87.

[15] [15a] C. H. Chang, F. H. Lin, T. F. Kuo, H. C. Liu, Biomed. Eng-App. Bas. C 2005, 17, 1; [15b] S. Sakai, H.Masuhara, Y. Yamada,T. Ono, H. Ijima, K. Kawakami, J. Biosci. Bioeng. 2005, 100, 127;[15c] C. S. D. Lee, J. P. Gleghorn, N. W. Choi, M. Cabodi, A. D.Stroock, L. J. Bonassar, Biomaterials 2007, 28, 2987; [15d] S. H.Chia, M. R. Homicz, B. L. Schumacher, E. J.-M. A. Thonar, J. Am.Coll. Surg. 2005, 200, 691;. [15e] H. A. Awad, M. Q. Wickham,H. A. Leddy, J. M. Gimble, F. Guilak, Biomaterials 2004, 25,3211.

[16] [16a] C. F. Degiorgi, R. A. Pizarro, E. E. Smolko, S. Lora,M. Carenza, Radiat. Phys. Chem. 2002, 63, 109; [16b] T. A.Becker, D. R. Kipke, T. Brandon, J. Biomed. Mater. Res. 2001, 54,76; [16c] S. Fujikawa, T. Yokota, K. Koga, Appl. Microbiol.Biotechnol. 1988, 28, 440.

011, 11, 22–35



www.mbs-journal.de

[17] [17a] N. F. Y. Tam, Y. S. Wong, C. G. Simpson, Biotechnol. Tech.1998, 12, 187; [17b] K. R. Reddy, K. Rajgopal, M. L. Kantam,Catal. Lett. 2007, 114, 36.

[18] [18a] L. Niklason, J. Gao, W. Abbott, K. Hirschi, S. Houser,R. Marini, R. Langer, Science 1999, 284, 489; [18b] B. Kim,J. Nikolovski, J. Bonadia, D. Mooney, Nat. Biotechnol. 1999, 17,979.




[19] [19a] M. Buschmann, Y. Gluzband, A. Grodzinsky, E. Hunziker,J. Cell. Sci. 1995, 108, 1497; [19b] C. Hunter, J. Mouw,M. Levenston, Osteoarthr. Cartil. 2004, 12, 117.

[20] [20a] T. C. Ezeji, N. Qureshi, H. P. Blaschek, J. Microbiol.Biotechnol. 2003, 19, 595; [20b] T. C. Ezeji, N. Qureshi,H. P. Blaschek, Appl. Microbiol. Biotechnol. 2004, 63,653.

11, 11, 22–35


Documents

Multi-Featured Macroporous Agarose–Alginate Cryogel: Synthesis and Characterization for Bioengineering Applications