Biosynthetic hydrogels—Studies on chemical and physical characteristics on long-term cellular...

Biosynthetic hydrogels—Studies on chemical and physicalcharacteristics on long-term cellular response for tissue engineering

Finosh Gnanaprakasam Thankam, Jayabalan Muthu

Sree Chitra Tirunal Institute for Medical Sciences and Technology, Polymer Science Division, BMT Wing,

Thiruvananthapuram 695 012, Kerala, India

Received 3 July 2013; accepted 15 July 2013

Published online 00 Month 2013 in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/jbm.a.34895

Abstract: Biosynthetic hydrogels can meet the drawbacks

caused by natural and synthetic ones for biomedical applica-

tions. In the current article we present a novel biosynthetic

alginate - poly(propylene fumarate) copolymer based

chemically crosslinked hydrogel scaffolds for cardiac tissue

engineering applications. Partially crosslinked PA hydrogel

and fully cross linked PA-A hydrogel scaffolds were prepared.

The influence of chemical and physical (morphology and

architecture of hydrogel) characteristics on the long term cel-

lular response was studied. Both these hydrogels were cyto-

compatible and showed no genotoxicity upon contact with

fibroblast cells. Both PA and PA-A were able to resist deleteri-

ous effects of reactive oxygen species and sustain the viabil-

ity of L929 cells. The hydrogel incubated oxidative stress

induced cells were capable of maintaining the intra cellular

reduced glutathione (GSH) expression to the normal level

confirmed their protective effect. Relatively the PA hydrogel

was found to be unstable in the cell culture medium. The PA-

A hydrogel was able to withstand appreciable cyclic stretch-

ing. The cyclic stretching introduced complex macro and

microarchitectural features with interconnected pores and

more structured bound water which would provide long-term

viability of around 250% after the 24th day of culture. All

these qualities make PA-A hydrogel form a potent candidate

for cardiac tissue engineering. VC 2013 Wiley Periodicals, Inc. J

Biomed Mater Res Part A: 00A:000–000, 2013.

Key Words: biosynthetic hydrogels, alginate-co-polypropyl-

ene fumarate copolymer, genotoxicity, macro and microarchi-

tecture, structured water and long term cell viability

How to cite this article: Thankam FG, Muthu J. 2013. Biosynthetic hydrogels—Studies on chemical and physical characteristicson long-term cellular response for tissue engineering. J Biomed Mater Res Part A 2013: 00A: 000–000.

INTRODUCTION

Hydrogels form one of the potent candidates for tissue engi-neering due to their ability to hold large amount of waterand their resemblance to native extra cellular matrix (ECM).These biomaterials can effectively grow the cells onto their3D network, traffic the nutrients, oxygen, and other aqueousmetabolites. Because of their close resemblance with theECM and similarity in the properties these are commonlyused for the engineering of organ or organ parts.1 Therebythese can meet the increasing demand for the organs andthe complications associated with transplantation. Severaltypes of hydrogels are being successfully employed for theengineering of various body parts like bones,2 cartilages,3

liver,4 pancreas,5 and so forth.Both natural and artificial hydrogels have been

employed in the in vitro designing of various organ parts.But the lack of sufficient mechanic strength and biostabilityforms a major obstacle in dealing with many of the compro-mising hydrogels.6 Natural polymer, alginate is extensivelystudied for tissue engineering applications due to its prom-ising biomimetic characters. But alginate fails in their loadbearing capability especially in an environment containing

monovalent ions.7 Synthetic biomaterials are mechanicallyrobust but their improper biodegradation profile and risk ofinflammatory responses offers a puzzle in using many ofthem in the de novo engineering of organ parts.8 If the bio-degradation products are proinflammatory it will alter thehost response significantly. This will lead to fibrotic capsulearound the implant which in turn inhibit the tissue remod-eling and function as a barrier for nutrient transport andangiogenesis.

Cardiac tissue engineering is a challenge because themain contractile element of the heart, the cardiomyocytes,are terminally differentiated and are unable to divide andrepair the lost part.9 The concentration of oxygen andnutrients at the infarct site will be very low due to theblockade of blood vessels, inflammatory reactions and thepresence of elevated concentrations of reactive oxygen spe-cies (ROS). This will affect the viability and the metabolicstatus of the cells seeded inside the hydrogels after implan-tation on the target site.10 So the hydrogels intended forcardiac tissue engineering applications should be able topromote the long term cell viability and function and willbe able to withstand this harsh environment even before

Correspondence to: J. Muthu; e-mail: [email protected] grant sponsors: Department of Science and Technology, New Delhi, Government of India; KSCST&E, Kerala, India

VC 2013 WILEY PERIODICALS, INC. 1

the seeded cells integrate with the host myocardium. Inaddition the hydrogel scaffolds should possess sufficientstrength to support myocardial wall at the infarct site,which was thinned due to the increased matrix metallo pro-teases (MMPs) activity.

The biosynthetic hybrid hydrogels can overcome thesedifficulties and have emerged as promising candidate as car-diac tissue engineering scaffolds. Biosynthetic hydrogels canexhibit structural, physiochemical, mechanical, and biologicalfunctionalities and controlled degradation profile.11,12 Severalcombinations of biosynthetic hydrogels have been studied forvarious biomedical applications like tissue engineering,13

drug delivery,14 gene delivery,15 and so forth. Biosyntheticglycidyl methacrylate and hyaluronic acid,16 poly(ethyleneglycol)–fibrinogen conjugates,17 poly(ethylene glycol) andheparin,18 poly vinyl alcohol, gluteraldehyde, chitosan, dex-tran, polyethylene glycol–chitosan, poly acrylic acid–alginate,chitin–PLGA, PAA–chitosan and PMAA–alginate were reportedfor various biomedical applications. Still we are lacking suffi-cient reports regarding the biofunctionally active biosynthetichydrogel scaffolds for cardiac tissue engineering applications.

The morphology and architecture of scaffolds for tissueengineering largely influence the cellular responses viz. celladhesion, viability, ingrowth, distribution, and the formationof extracellular matrix.19,20 The cell-responsive chemical ele-ments present in a hydrogel polymer also largely influencethe cellular responses, that is, binding of cells as well asdegradation by cells. The lack of cell-responsive featureslimits the cell proliferation and elongation, biomolecule pro-duction and production of highly ordered structures.21 Cellresponsive features are introduced with different chemicalfunctional groups and physical morphology of the hydrogels.Therefore for cardiac tissue engineering, it is a greatendeavor to develop biofunctional hydrogels to perform itsintended mechanical function without compromising thebiological function. The present article deals with the co-polymer hydrogel based on the cellular response of biosyn-thetic natural polysaccharide alginate and synthetic polyes-ter poly propylene fumarate influenced by chemical andphysical (morphology and architecture of hydrogelscaffolds) characteristics for cardiac tissue engineeringapplications.

MATERIALS AND METHODS

Preparation of biosynthetic hydrogelHydroxyl terminated-poly (propylene fumarate) (HT-PPF)oligomer was synthesized using maleic anhydride and 1,2-propylene glycol as per the procedure described else-where.22 Poly (propylene fumarate)-alginate copolymer(PPF-AG) was prepared by the acid catalyzed blending ofsodium-alginate powder (guluronic acid (39%) and mannu-ronic acid (61%) from brown algae, medium viscosity pur-chased from Sigma–Aldrich, USA, Product No.A2033) at70�C in the ratio 1:2 (PPF:Alginate). The solid mass soformed was then slowly dissolved in minimum distilledwater and stored at room temperature.

From this copolymer, two hydrogel scaffolds, PA (partiallycrosslinked) and PA-A (fully crosslinked) were prepared by

solvent casting followed by freeze drying. The PA was pre-pared by crosslinking only the alginate fraction of the PPF-AG(60 g) with Ca21 ions by incubating 10% CaCl2 for 4 h atroom temperature. The PA-A was prepared by cross linking3.75 g acrylic acid with 60 g PPF-AG copolymer resin. Thealginate fraction was then crosslinked with Ca21 ions assame as PA hydrogel. The hydrogels so formed were thenimmersed in distilled water at room temperature for 24 h forthe leaching out of unreacted molecules. The hydrogel sheetswere cut (4 cm 3 1.4 cm) and swelled in distilled water. Thesamples were then freeze dried for 3 h, sterilized using ethyl-ene oxide and used for biological evaluation.

In vitro evaluation of cell viability of peripheral bloodmononuclear cells (PBMCs)Fresh peripheral blood mononuclear cells (PBMCs) wereisolated from the blood of healthy human volunteers byadding to volume of HiSep (HIMEDIA) without hemolysisand centrifuged at 2000 rpm for 20 min as per the manu-facturer’s instructions. Then the PBMC layer was removedand washed with PBS and resuspended in RPMI-1640medium supplemented with 20% heat inactivated FBS andantibiotics (Penicillin and Streptomycin). The hydrogels (1-cm diameter) were extracted in 1-mL RPMI-1640 mediumfor 24 h at 37�C in a CO2 incubator. This extract was usedto seed �2 3 104 PBMCs. Then the viability was deter-mined by MTT23 and neutral red (NR)24 assay after 72 h toevaluate the viability response of the hydrogel.

In vitro evaluation of genotoxicity of hydrogelsThe genotoxicity of PA and PA-A hydrogels were evaluatedby comet assay on L929 cells. L929 fibroblast cells werecultured in basic medium composed of Dulbecco’s ModifiedEagle’s Medium with glucose (HiMedia), supplemented with10% fetal bovine serum (FBS), Penicillin, streptomycin, andsodium bicarbonate. The medium was replaced with freshmedium once in 3 days and the cells were subcultured afterattaining around 70% confluence.

To confirm the absence of genotoxic effects of the hydro-gel upon contact with the cells, the DMEM-swelled hydro-gels were placed over a subconfluent layer of L929fibroblasts and incubated at 37�C for 24 h. These cells werethen subjected to comet assay as per the following proce-dure. The microscope slides were precoated with 1mL of0.75% normal melting point agarose (NMA) and stored at4�C. This layer was removed before use and 120 lL of0.75% NMA was pipetted on to the slides, which were thencovered with cover slips. Cells were trypsinized, mixed with50 lL of low melting point agarose and pipetted over thefirst layer of agarose. NMA (80 lL) was used as a final pro-tective layer. Slides were placed in cold lysing solution(2.5M NaCl,100 mM Na2EDTA, 10 mM Tris pH 10 and 1%SDS to which 10% DMSO and 1% Triton X 100 were addedimmediately prior to use) for overnight at 4�C. After lysisslides were placed in electrophoresis buffer (300 mM NaOHmM and Na2EDTA PH13) for 20 min to allow unwinding ofDNA. Then electrophoresis was conducted in the samebuffer by applying an electric current of 0.8 V cm21 (300

2 THANKAM AND MUTHU BIOSYNTHETIC HYDROGELS

mA) for 15 min. Finally slides were washed in neutraliza-tion buffer (0.4 lL Tris, pH 7.5) three times for 5 min andstained with 50-lL ethidium bromide (20 lg mL21). Theexcess stain was washed with PBS and viewed under fluo-rescent microscope using green filter. The extent of DNAdamage was quantified by the comet reading software Tri-Tek cometScore Freeware 1.6.1.13 with respect to a controland H2O2 treated cells (200 mM H2O2 was added to the cul-ture and incubated for 24 h).

In vitro evaluation of free radicals scavenging responseof hydrogelThe free radical scavenging effect of hydrogel scaffolds werealso investigated on L929 fibroblast cell line. An oxidativestress was induced onto the cell culture with the addition of200 lM H2O2.

25 Then hydrogel scaffolds (1cm diameter)swelled in DMEM was placed on the stress-induced cell layerand incubated at 37�C for 24 h. Then the viable cells weremicroscopically evaluated by live-dead assay. Cultures withH2O2 alone (without scaffolds) and cells alone (without scaf-folds and H2O2) were also carried out as controls. The live–dead assay was conducted using a mixture of acridine orange(100 lg mL21) and ethidium bromide (100 lg mL21) andviewed immediately under an epifluorescence microscope(Optika SRL) using blue filter for acridine orange and greenfilter for ethidium bromide.26 Two images taken from thesame field were merged by Photoshop8 CS software.

The intra cellular glutathione (GSH) level was determinedmicroscopically. The hydrogel scaffolds were removed andcells were washed with PBS. Then 20 lL monochlorobimane(10 lM in methanol) was added and incubated at room tem-perature for 30 min.The excess stain was removed by PBSwashing. The cells were then imaged by the fluorescentmicroscope at blue filter without changing the settings of themicroscope. The intensity of the images were quantified bythe Image J software and compared with that of controls.27

Studies on the effect of cyclic stretching of hydrogel oncellular responseThe water swollen DMEM stable PA-A hydrogel was subjectedto accelerated cyclic stretching for around 15,000 cycles (1/4thcycles of the complete fatigue life of PA-A hydrogel) in waterbath at 37�C. The stretching was carried out with frequency 6cycles s21, amplitude 2.5 mm and load 0.91N using a biome-chanical tester (M/S Test Recourses USA). The samples werethen freeze dried for 3 h and used for further analysis.

Water content and swelling ability of stretched PA-AhydrogelThe freeze dried samples were soaked in distilled water for24 h at room temperature to attain maximum swelling. Theswelled sample was again weighed in an electronic weighingbalance. The equilibrium water content and the swelling effi-ciency of the hydrogels were calculated using the equations.

EWC ¼ ðWeight of the swollen hydrogel2Weight of the dry hydrogelÞWeight of the swollen hydrogel

3100

%Swelling ¼ ðWeight of the swollen hydrogel2Weight of the dry hydrogelÞWeight of the dry hydrogel

3100

Evaluation of surface morphology of PA-A before andafter stretchingThe pore morphology was examined by environmental scan-ning electron microscopy (ESEM) analysis prior and poststretching. Freeze dried hydrogels (2 3 1 cm2) was viewedunder low vacuum condition.

Determination of phase transition of water in PA-Ahydrogels by DSC analysisThe phase transitions of water in the hydrogel after cyclicstretching were determined with by differential scanningcalorimetry (DSC) as per ASTM 537-07 method. The water-swollen hydrogel (10 mg) was first cooled to 270�C andthen heated to 100�C at the rate of 5�C min21 in nitrogenatmosphere. The heating process was recorded.

Determination of long-term cell growth on stretchedPA-A hydrogelThe cellular response and long term growth on stretchedPA-A hydrogel was assessed using L929 cells. A modified

version of MTT assay was adopted.28 Around 5 3 104 cellswere seeded to the preincubated PA-A on a 24-well plateand allowed to proliferate for 24 days. At regular intervalsthe hydrogel was washed with PBS and incubated in 1 mLMTT solution (1 mg mL21) at 37�C for 3 h. After incubationthe hydrogel was extracted with the solution containing0.01N HCl in isopropanol. The contents were then vortexedfor 10 min to detach all the cells adhered in the hydrogel. Itwas then kept for 30 min and centrifuged at 5000 rpm for10 min to settle the hydrogel and cell debris. Then the ODof the supernatant was measured at 570 nm. A control wasset up with cells grown without hydrogel. A blank contain-ing hydrogel without cells was also treated in a similarmanner. From the OD values the % viability of the cellsgrown inside the hydrogels were calculated.

Statistical analysisAll experiments consisted of five or six samples from eachgroup. The values are presented as means 6 standard

ORIGINAL ARTICLE

JOURNAL OF BIOMEDICAL MATERIALS RESEARCH A | MONTH 2013 VOL 00A, ISSUE 00 3

deviations. Statistical analysis was done with one wayANOVA using online calculator, Statistics Calculator version-3 beta and the level of significance was set at p < 0.05 forall calculations.

RESULTS AND DISCUSSIONS

Evaluation of in vitro cell viability of peripheral bloodmononuclear cells (PBMCs)Hybrid biosynthetic hydrogels have emerged as promisingbiomaterials for tissue engineering applications due tofavorable physicochemical, mechanical properties and bio-compatibility. The present hydrogels with PPF and alginateunits have been prepared as copolymer (PPF-AG) whosemolecular weight was 784 (Mn) and 2010 (Mw) and polydis-persity 2.56. The alginate fraction of PA was alone crosslinked with Ca21 to form partially crosslinked PA hydrogelscaffold. With fully crosslinked PA-A hydrogel, both alginateand PPF portion was cross linked. PPF portion was crosslinked by the vinyl monomer acrylic acid. The chemical con-stituents of these hydrogels are shown in Figure 1.

The response of hydrogel extracts on the viability ofPBMCs were determined by MTT and NR assay. The viabilityof PBMCs can be indirectly correlated with the occurrenceof inflammatory responses, since the apoptotic or necrotic

cell death of these leukocytes were reported to elicit inflam-mation.29 The mild and acute inflammation immediatelyafter implantation and the biodegradation (if the degrada-tion products are inflammatory) will alter the host responsesignificantly. Even a slight inflammation may lead to fibroticcapsule around the implant which may inhibit tissue remod-eling and function as barrier to the transport of nutrientand angiogenesis.

Therefore evaluation of inflammatory and genotoxicresponse of hydrogel intended to use in biomechanical envi-ronment is essential. PBMCs represent major components ofthe immune system and they play a key role in acuteinflammation due to the chemokine secretions. The progres-sion of inflammation involves necrosis of these inflamma-tory cells and the extra cellular release of proapoptotic andhistotoxic factors which will results in considerable reduc-tion of the viable PBMCs. In short the higher rate of apopto-tic death of PBMCs was attributed to the inflammatoryresponse of the hydrogel extracts as reported elsewhere.29

The inflammatory response can also be confirmed by theexpression of surface biomarkers like CD11 and the expres-sion and release of inflammatory biomarkers like TNF alphaor IL1b on PBMCs. The present hydrogels exhibit more than80% viability of cells (Fig. 2). Therefore it can be concluded

FIGURE 1. Chemical constituents of PA and PA-A hydrogels. [Color figure can be viewed in the online issue, which is available at

wileyonlinelibrary.com.]


that the degradation products or the leached particles fromthese hydrogels are nontoxic to the immune cells and maynot initiate their activation and subsequent immune reac-tions. This can be extrapolated to various cell types of car-diac tissues and infarcted myocardium where the possibilityof PBMC accumulation is far greater.

Evaluation of in vitro genotoxicity of L929 cells oncontact with hydrogelsThe mutagenicity of a scaffold depends not only on chemicalproperties but also on physical characteristics like porosity,hardness, surface energy, and so forth.30 The comet assay orsingle cell electrophoresis is widely used for quantification ofgenotoxicity. In the present studies, the cells were incubatedwith the scaffold materials and then subjected to comet assayto check the genotoxic effect of compounds leaching from it,because in culture medium or biological fluids, the presenceof higher amount of Na1 and other monovalent ions maycause a reversible exchange of the cross linked Ca21 withmonovalent ions. This will result in a slight unwinding of thecross linked alginate fraction of the hydrogels which in turndisturb the arrangement of other polymer chains and the co-monomers and cross linkers used. So the chances of forma-tion and leaching of the degradation products and unreactedreactants will be higher immediately after the exposure ofthese hydrogels to culture medium. The medium extractedwith PA and PA-A hydrogel samples were tested for theirability to maintain the genetic integrity by comet assay. As

per the assay both the PA and PA-A hydrogel samples showedvalues of the genotoxic parameters very close to that of thecontrol cells. For both the samples, DNA in the head region ismore than 99% and in the tail region it is less than 1%. Thediameter of the head region was also maximum (Table I, Fig.3). The studies reveal these hydrogel scaffolds are capable ofmaintaining the genetic integrity without altering the genomeof the cells.

Evaluation of free radicals scavenging response ofhydrogelThe cell-responsive elements present in a hydrogel polymerlargely influence the cellular responses, that is, binding ofcells as well as degradation by cells. The lack of cell-responsive features limits the cell proliferation and elonga-tion, biomolecules production and the synthesis of highlyordered structures.21 Cell responsive features are intro-duced with different chemical functional groups of thehydrogels. The present hydrogels comprise PPF and alginateunits with different degree of crosslinking and functionalgroups (acidic carboxyl, hydroxyl, ester, ether, methyl groupsand unsaturated double bonds).

The scavenging effects of PA and PA-A hydrogels wereassessed by live/dead assay and intracellular GSH staining.Live dead assay by acridine orange/ethidium bromidereveals the apoptotic status of the cells and displays viablehealthy cells, viable early apoptotic cells, non viable lateapoptotic cells and nonviable necrotic cells due to the differ-ence in their emitted fluorescent color. As per the assay thenormal viable cells display a uniformly stained greennucleus, early apoptotic cells have a highly condensed orfragmented green nuclei while the late apoptotic, necroticand nonviable cells have orange nuclei due to ethidium bro-mide intercalation.31 The images from the live/dead assayshowed that a greater number of healthy viable cells werepresent in the H2O2 loaded-hydrogel scaffolds when com-pared with the H2O2 treated control, where most of the cellswere found to be apoptotic (Fig. 4). The mean gray value ofmonochlorobimane stained intra cellular GSH was higher inH2O2 loaded-hydrogel scaffolds when compared with theH2O2 treated control (Table II, Fig. 5). The fluorescentimages of cells stained with monochlorobimane are shownin Figure 6. The images show better GSH expression inhydrogel-treated samples. This showed that the presence ofthe hydrogels in the culture reduced the free radicalinduced toxicity to the cells. But the mechanism of this pro-tective effect is yet to be determined.

The reactive oxygen species (ROS) has both structuraland functional effects on MI and postmyocardial remodeling.

FIGURE 2. Viability assays PBMCs on the medium extracted with PA

and PA-A hydrogels showing viability more than 80%. [Color figure

can be viewed in the online issue, which is available at


TABLE I. Evaluation of Genotoxicity of PA and PA-A Hydrogels by Comet Assay

Samples (n 5 30)Head Diameter(Px) (p < 0.01)

% DNA in Head(p < 0.001)

Tail Length (Px)(p < 0.001)

% DNA in Tail(p < 0.001)

Control 20.51 6 4.58 99.72 6 0.38 0.76 6 1.36 0.36 6 1.38H2O2 29.5 6 13.16 70.37 6 9.87 10.75 6 5.5 29.62 6 9.87PA 25.42 6 7.82 99.84 6 0.3 1 6 0 0.15 6 0.3PA-A 24 6 6.9 99.38 6 0.47 0.33 6 0.48 0.5 6 1.53

ORIGINAL ARTICLE


FIGURE 4. Free radical scavenging effect of hydrogels showing the presence of healthy cells seeded in presence of PA (C) and PA-A (D) in com-

parison with control (A) and H2O2 treated cells (B). The relative higher green colored cells in the hydrogel-treated samples showing viable cells

when compared to that of H2O2 treated cells. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

FIGURE 3. Evaluation of genotoxicity of hydrogel scaffolds by comet assay showing the representative images of control (A), H2O2 treated (B),

PA treated (C) and PA-A treated (D). [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]


ROS will induce hypertrophy and apoptosis to the survivingand unaffected cells of the heart after the infarct. Theinflammation and the associated mechanisms at the infarctsite also will leads to the increase in ROS concentrations atand adjacent sites of infarction. Moreover ROS can activatethe expression of matrix metalloproteases (MMP) in cardiacfibroblasts and other cells. MMPs will enhance the cardiacremodeling process and activate fibrosis and scar forma-tion.32 So the arresting of propagation of ROS with scaffoldsat the MI site can resist the remodeling and the associatedcomplications.

Glutathione (GSH) is a tripeptide that exists in two inter-convertible oxidized (GSSG) or reduced (GSH) states ofwhich the GSH predominates in the cells under normalphysiology. They act as reducing equivalents for a wide vari-ety of reactions especially to the enzymes for the detoxifica-tion of ROS. GSH is regarded as a marker for the healthycells and its depletion will impose apoptosis. So the higherGSH level in the cells is an indication of apoptosis resistancewhile the oxidative stress is a potential inducer of the same.In short there is an inverse relation between the oxidativestress and intracellular GSH level. To relieve the oxidativestress the normal antioxidant defense mechanism of the

cells will be activated and their utilization of GSH will leadto conversion to their oxidized form, GSSG. If the oxidativestress persists inside the cells, the reconversion of GSSG toGSH will be reduced or delayed leading to the oxidativedamage.33 In our case the presence of hydrogels in the cul-ture caused a reduction in the apoptosis and increase in thecellular GSH level. This signifies the oxidative resistancethat can be offered by the hydrogel scaffolds, especiallyPA-A, in a hostile environment like infarct site.

Effect of cyclic stretching of hydrogel on cellularresponseThe present hydrogels were tested for their stability inDMEM. PA hydrogel was unstable in DMEM with completedissolution. But PA-A hydrogel was found to be stable even

FIGURE 5. Comparison of the gray value of intra cellular GSH staining

on cells seeded along with PA and PA-A hydrogel scaffolds. [Color

figure can be viewed in the online issue, which is available at


FIGURE 6. Fluorescent images of cells stained with monochlorobimane for GSH showing better GSH expression. Control (A), H2O2 treated (B),

PA (C) and PA-A (D). All the images were taken at 403 magnifications without changing the settings and adjustments of the microscope. These

images were converted to 8bit gray scale for measuring mean gray value by ImageJ software. [Color figure can be viewed in the online issue,

which is available at wileyonlinelibrary.com.]

TABLE II. Comparison of the Gray Value of Intracellular GSH

Staining on Cells Seeded Along with PA and PA-A Hydrogels

Sl no. Samples Mean Gray Value

1 Control 55.96 6 3.572 H2O2 treated 45.87 6 1.73 PA 52.93 6 1.084 PA-A 58.73 6 3.22

ORIGINAL ARTICLE


after 2 weeks. The lower stability of PA hydrogel was dueto the exchange of Ca21 with monovalent ions from themedium which increases the water permeability that resultsin more Ca21 exchange where as in PA-A the extensive crosslinking of fumarate groups of PPF will mask the Ca21 crosslinked alginate fraction. This will prevent the monovalentions to access the Ca21 for exchange which imparts stabilityto the PA-A hydrogel scaffolds.

The DSC heating curve of PA-A hydrogel after cyclicstretching displayed endothermic peak due to the melting offrozen water. The sharp endothermic peak has appearedwith the onset of transition at 21.39�C for PA-A hydrogel(Fig. 7). This is due to the melting of cumulative frozenwater (Wf) consisting freezing free (Wff) and freezing bound(slightly structured water) (Wfb) as reported by Xianget al.34 The freezing water content (Wf) was calculated fromthe enthalpy of melting endotherm and enthalpy of meltingof pure water (334 J g21) using the equation Wf 5

(DHEndo/DHw) 3 100, where DHEndo is the enthalpy of melt-ing of frozen water in the hydrogel, DHw enthalpy of meltingof frozen pure water (334 J g21). The non freezing boundwater content (Wnb) was calculated from the differencebetween the equilibrium water content (EWC) and freezingwater content (Wf). The equilibrium water content (%)(EWC) and swelling (%) of PA-A hydrogel was found to be62.82 6 2.46 and 169.98 6 18.98, respectively. PA-A hydro-gel after cyclic stretching exhibits freezing water content(Wf) and nonfreezing bound water content (Wnb) as 35.3and 27.52%, respectively. Xiang et al.34 have reported thathigher the equilibrium water content (EWC) higher is thefreezing water content (Wf).

The scaffolds used for tissue engineering were fabricatedby conventional methods as expanded grafts, textile weaves,porous films, and sponges. Invariably these scaffolds possesssimple macroarchitecture and homogeneous microstruc-tures. The pore size and void fraction in any scaffoldinfluence the cellular attachment, proliferation and ECMdeposition. For successful scaffold to integrate with host tis-

sue and function biomechanically, it should have complexmacro and microarchitectural feature combined with cell-responsive elements from the polymer. The optimum poresize for neovascularization is reported to be 5 lm, fibroblastinfiltration requires 5–15 lm, and around 500 lm for fibro-vascular tissue. The oxygen tension is low beyond 200 lmfrom the blood vessel which affect the cell growth. The poreinterconnectivity is another factor dealing with the viabilityof the cells inside the scaffolds.35 The pore morphology andmicrostructure of PA-A hydrogel after cyclic stretching were

FIGURE 7. DSC thermogram of heating curves showing melting of freezing free water (endothermic peak) in PA-A hydrogel after cyclic stretch-

ing. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

FIGURE 8. ESEM analysis of PA-A hydrogel showing the changes in

surface morphology of stretched (A) and unstretched samples (B).


determined by ESEM analysis. ESEM analysis revealed thepresence of complex macro and microarchitectural featurewith interconnected pores on the surface (Fig. 8) as well asin the core. The PA-A hydrogel showed a good porosity andinterconnectivity in the surface which is the reflection of itsinternal architecture.

The long-term cell viability and cell penetration instretched hydrogel scaffold were assessed. The cell viabilitywas quantified for 24 days and a progressive increase inviability was found in the cells seeded onto the scaffoldsindicating the cells would have directed towards the innernetworks of the hydrogel. This porosity enhances the cellviability and metabolic activity which showed the greaternumber of viable cells inside the hydrogel scaffolds (Fig. 9).The long term cell viability is also attributed greatly to thepresence of structured water to an extent of 30% alongwith slightly structured water and free water in the struc-tural networks of the hydrogel. The newly induced poresand structured water can ensure the uninterrupted cyclingof nutrients and metabolites throughout the bulk of PA-Ahydrogels thereby offers a long term residence to theseeded cells.

CONCLUSION

Biosynthetic hydrogel scaffolds were prepared from poly(-propylene fumarate)-co-alginate copolymer. Limited cross-linking with Ca21 produced partially crosslinked hydrogel(PA). Crosslinking with Ca21 and acrylic acid produced fullycrosslinked hydrogel (PA-A). Both the scaffolds were cyto-compatible, nongenotoxic and were able to resist the ROSattack to a greater extent. But PA hydrogel was unstable inthe culture medium which limits its use for only short termapplications. The cyclic stretching of PA-A hydrogel scaffoldintroduced structured water bound with polymer and com-plex macro and microarchitectural features with intercon-nected pores which provides sufficient room for the cells topenetrate inside which will impart long term viability andfunction. The absence of inflammatory response and geno-toxicity, capability to scavenge free radical, long term viabil-ity of cells and growth in stretched PA-A hydrogel scaffoldreflects the potential application for cardiac tissueengineering.

ACKNOWLEDGMENTS

The authors thank Prof. K. Radhakrishnan, Director, SCTIMSTfor providing the facilities to carry out this work.

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