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International Journal of Biological Macromolecules 49 (2011) 1131– 1136

Contents lists available at SciVerse ScienceDirect

International Journal of Biological Macromolecules

jo u r n al hom epa ge: ww w.elsev ier .com/ locate / i jb iomac

ytotoxicity and mechanical behavior of chitin–bentonite clay basedolyurethane bio-nanocomposites

halid Mahmood Ziaa, Mohammad Zubera,∗, Mehdi Barikanib, Rizwan Hussainc, Tahir Jamild,ohail Anjuma

Institute of Chemistry, Government College University, Faisalabad 38030, PakistanDepartment of Polyurethanes, Iran Polymer and Petrochemical Institute, Tehran, IranP.O. Box 2216, NESCOM, Islamabad, PakistanDepartment of Polymer Engineering and Technology, University of the Punjab, Lahore, Pakistan

r t i c l e i n f o

rticle history:eceived 9 August 2011eceived in revised form 5 September 2011ccepted 11 September 2011vailable online 16 September 2011

a b s t r a c t

Chitin based polyurethane bio-nanocomposites (PUBNC) were prepared using chitin, Delite® HPS ben-tonite nanoclay enriched in montmorillonite (MMT), 4,4′-diphenylmethane diisocyanate (MDI) andpolycaprolactone polyol CAPA 231 (3000 g/mol−1). The prepolymers having different concentration ofDelite HPS bentonite nanoclay were extended with 2 moles of chitin. The structures of the resulted poly-mers were determined by FT-IR technique. The effect of nanoclay contents on mechanical properties and

eywords:hitinolyurethaneio-nanocompositesentonite clay

in vitro biocompatibility was investigated. The mechanical properties of the synthesized materials wereimproved with increase in the Delite HPS® bentonite nanoclay contents. Optimum mechanical propertieswere obtained from the PU bio-nanocomposite samples having 4% Delite HPS® bentonite nanoclay. Theresults revealed that the final PU bio-nanocomposite having 2% Delite HPS® bentonite nanoclay contentsis ideal contenders for surgical threads with on going investigations into their in vitro biocompatibility,

ical p

yto-toxicity non-toxicity, and mechan

. Introduction

Polyurethanes have potential array of commercial applicationss they can be molded, injected, extruded and recycled [1]. Molec-lar characterization and morphological studies of polyurethanelastomers (PUEs) have been reported by many researchers. Theffects of the diisocyanate structure [2] and length of chain exten-er (C.E.) using �,�-alkane diols on the crystallinity, surfaceorphology [3] and thermo-mechanical properties [4] of PUEs

ave been investigated and well documented. Detailed molecularharacterization [5], XRD studies with blends of chitin/1,4-butaneiol (BDO) [6,7] and varying diisocyanates structure [8] have beeneported in the literature. The thermal [5] and shape memory prop-rties [9] of chitin-based polyurethane elastomers have also beenreviously discussed and reported. The physical and elastic prop-rties of PUs developed for durable application are much superiorver other competitors mentioned above. Of the developed PUs, thever-whelming majority are biostable materials, designed to with-

tand in service for long periods of time [10]. One of the intendedses of these polymers is the insertion in a living organism forxtended period; therefore a great care has to be taken in the choice

∗ Corresponding author. Tel.: +92 321 6682375; fax: +92 41 9200671.E-mail address: mohammadzuber@gmail.com (M. Zuber).

141-8130/$ – see front matter © 2011 Elsevier B.V. All rights reserved.oi:10.1016/j.ijbiomac.2011.09.010

roperties.© 2011 Elsevier B.V. All rights reserved.

of their building blocks. Their degradation products have to be bio-compatible, non-toxic and metabolized or eliminated by the livingorganism [11].

Polymer nanocomposite is a class of hybrid materials composedof an organic polymer matrix with dispersed inorganic nano-fillers, which have at least one dimension in nanometer range[12]. Polymer nanocomposites can commonly be obtained by eitherthe sol–gel method or the intercalation method [13]. The fourmain strategic processes for preparing polymer/layered silicatenanocomposites are exfoliation–adsorption, in situ intercalativepolymerization, melt intercalation and template synthesis [14].

Biostability and biocompatibility of poly(ester urethane)[15] poly(3-hydroxybutyrate-co-3-hydroxyvalerate-co-3-hydroxyhexanoate) [16] have been discussed in the literature.Some references are also available on the study of biocompatibilityand bioactivity of plasma-treated biodegradable poly(butylenesuccinate) [17]. Synthesis, mechanical properties, biocompati-bility, and biodegradation of polyurethane networks from lysinepolyisocyanates have also been cited [18]. It is considered thatthe introduction of polysaccharides, e.g., chitin as chain exten-der/crosslinker; a part of hard segment can enables to get non-toxic

and biocompatible polyurethane product. In our previous stud-ies [19,20] biocompatibility of PU based on chitin/BDO blendswere investigated. In its continuation, the present article reportsthe effect of diisocyanate structure on the biocompatibility and

1132 K.M. Zia et al. / International Journal of Biological Macromolecules 49 (2011) 1131– 1136

Table 1Sample code designation and different formulation of PU bio-nanocomposites (molar ratio (as whole) of CAPA231/MDI/chitin; 1.0:3.0:2.0).

Sample code Polyol CAPA231a

(0.0156 mole)Diisocyanate MDIb

(0.047 mole)Chain extender/crosslinkerChitin (0.0313 mole)

Bentonite nanoclay %by mass

PUBNC1c PCLd; (31.25 g) MDI; (11.95 g) Chitin; (4.25 g) 0.0%PUBNC2 PCL; (31.25 g) MDI; (11.95 g) Chitin; (4.25 g) 1.0%PUBNC3 PCL; (31.25 g) MDI; (11.95 g) Chitin; (4.25 g) 2.0%PUBNC4 PCL; (31.25 g) MDI; (11.95 g) Chitin; (4.25 g) 4.0%PUBNC5 PCL; (31.25 g) MDI; (11.95 g) Chitin; (4.25 g) 8.0%

a Trade name of the used polyol (molecular weight 3000 g/mol).

mSnCeptobat

2

2

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b 4,4′-Diphenylmethane diisocyanate.c Polyurethane bio-nanocomposites.d Polycaprolactone polyol.

echanical behavior of chitin-based polyurethane elastomers.ynthesis and XRD properties of chitin based polyurethane bio-anocomposites have also been reported and discussed [21,22].onsidering versatile properties of PU and wound healing prop-rties of the chitin, it is considered that resulting product will beroved to be suitable candidate for biomedical implants. Due tohe unique and novel characteristics of chitin, this article reportsn the biocompatibility evaluation of chitin/bentonite nanoclayased polyurethane bio-nanocomposites. In view of the futurepplication of the synthesized PUs in the biomedical field, theiroxicity is currently being investigated and reported.

. Experimental

.1. Materials

Chitin was procured by the Sigma–Aldrich chemicals andt was further purified according to the established methods

Fig. 1. Synthetic route for the synthesis of Delite HPS bentonite nano

[23,24]. Delite® HPS bentonite (inorganic MMT with highest cationexchange capacity (CEC) i.e., 74 meq/100 g) was provided by LaviosaChemica Mineraria (LCM) SpA, Italy. Bentonite nano-clay, 4,4′-diphenylmethane diisocyanate (MDI) and polycaprolactone polyol,CAPA 231 (3000 g/mol−1) were used in this study.

2.2. Synthesis of polyurethane bio-nanocomposites

For this investigation weighed amount of clay (Table 1) wasthoroughly dispersed for 1 h at 70 ◦C with the solvent i.e., dimethylsulfoxide (DMSO), and then polyol was mixed to the clay solventmixture with continuous stirring for about 1 h at 90 ◦C. Bentonitenanoclay based PU prepolymer was formed following the addi-

tion of MDI and lasting the reaction at 100 ◦C for about another1 h at 100 ◦C. The polymerization was completed by adding thechitin at last step as chain extender/crosslinker. When color homo-geneity was obtained in the reactant mixture, the dispersion of

clay-chitin-based polyurethane bio-nanocomposites (PUBNC).

K.M. Zia et al. / International Journal of Biologic

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ig. 2. FT-IR spectra: (a) chitin based PU-prepolymer with bentonite clay and (b)hitin based PU-prepolymer without bentonite clay.

hain extender was considered complete, and the liquid polymeras cast onto a Teflon material to form a uniform sheet 2–3 mm

hick. The synthesized polymer was first placed under vacuumo remove the solvent from the synthesized bio-nanocompositesnd then cured in a hot-air circulating oven at 100 ◦C for 24 h.he cured sample sheets were then stored for 1 week at ambientemperature (25 ◦C) and 40% relative humidity before testing. Its worthwhile mentioning that the solvent was used for the dis-ersion of bentonite clay in all the samples even into the samplehere bentonite nanoclay was not used, for the purpose to make a

omparative study. A schematic illustration of the chemical routeor synthesis of bentonite nanoclay-chitin based polyurethane bio-anocomposites (PUBNC) is shown in Fig. 1.

.3. Characterization

Infrared measurements were performed on a Bruker-IFS8 Fourier Transform Infrared (FT-IR) Spectrometer (Ettlingen,ermany). Mechanical properties including tensile strength, mod-lus and elongation at break were determined from stress–strainurves with MTS tensile tester model 10/M at a strain rate of0 mm/min. The measurements were performed at 25 ◦C with alm thickness of about 1 mm and stamped out with an ASTM D-38 die. The hardness of all the samples was measured by a Zwick

nstrument in shore A scale. All the data presented were averagef five replicates. Cell culture assay was employed following theethod reported in our previous studies [19,20].

. Results and discussion

.1. Structural characterization

FT-IR spectra of polyurethane (PU) prepolymer with nanofillerbentonite clay) and PU prepolymer without nanofiller are pre-ented in Fig. 2. The FT-IR spectra of the PU prepolymers showed

very strong, new peaks at about 1728 cm−1 (a) and 1725 cm−1(b)hich were assigned to C O stretching of soft segment of PCL.nother new peak was also observed at about 2255 cm−1 whichas assignable to isocyanate (–NCO) group. Second new peakas also observed at about 1460 cm−1 which was assignable to

rethane –NH group. To provide clear information about the vibra-ional mode changes due to involvement of bentonite clay intohe polyurethane backbone, the FT-IR spectra of PU prepolymerith nanofiller (Delite HPS® bentonite clay) and without nanofiller

al Macromolecules 49 (2011) 1131– 1136 1133

were compared. It is obvious from Fig. 2(a) and (b) that boththe spectra have shown almost a complete set of peaks at thesame positions. Furthermore, it was observed that the intensityof hydrogen-bonded-NH increased as the chitin content increased,suggesting that the hydrogen bonds of chitin were broken and newhydrogen bonds between chitin and NCO terminated PU prepoly-mer were formed [21]. Based on these results of FT-IR spectrum,it was thought that chitin had already effectively crosslinked withpolyurethane, which greatly affected on the properties of the PUbio-nanocomposites.

Optical microscope (OM) is a powerful technique to monitor theformation and microstructure of intercalated clays. The existence ofthe intercalated clay by PU in the hybrid was confirmed by OM [21],as OM is necessary to determine the nature of the nanocompositesand to provide additional information that will be helpful in theinterpretation of the results. It is obvious from the results of OMphotographs that bentonite nanoclay is fully dispersed in all theprepared samples and presence of the clay contents is clearly visibleon the surface of the samples. Based on the above OM results, thedirectly casted chitin–bentonite clay based PU sample is apparentlya bio-nanocomposite with intercalated structure and disorderedlyexfoliated structure of bentonite clay.

3.2. Evaluation of cytotoxicity and cytocompatibility

Cell culture tests were used to evaluate both cyto-toxicity andcyto-compatibility of the specimens. In the cell culture method,the performance of a cell is investigated by comparing it with acontrol. A control (polystyrene tissue cell culture) is a sample thor-oughly compatible with cells and is cultured with the main samples.A material is considered to be biocompatible, if it supports cellattachment and growth. The cytotoxicity of the degradation prod-ucts was measured by live/dead staining. Representative live/deadimages of L-929 fibroblast cells incubated for 48 h on tissue culturepolystyrene in degradation products from each of the five PU bio-nanocomposite samples are shown in Fig. 3. L-929 fibroblast cellsseeded on the PU bio-nanocomposite samples show some poor via-bility, suggesting that the PU bio-nanocomposites are toxic. Opticalphotomicrographs of L-929 fibroblast cell interaction with the con-trol, PUBNC1, PUBNC2, PUBNC3, PUBNC4 and PUBNC5 are shownin Fig. 3. The evaluation of cell morphology shows that cells sur-vived and grew on the bottom of the wells of samples PUBNC1 andPUBNC2 with different rates, attributed to the amount of the DeliteHPS clay contents with spindle shape morphology. Furthermoreone of these two samples i.e., PUBNC1 appeared to give off sometoxic or inhibitory leachate, since round shaped cells also grew toconfluence in their wells along with spindle shape cells. It is clearfrom Fig. 3 that round shaped cells are visible even on the sur-face of PU bio-nanocomposites film which provides evidence thatthe synthesized material have surely toxic characteristics and thistoxic character increased with increase in bentonite clay contents.In comparison with PUBNC1 and PUBNC2 the former showed somebetter results than the later. This behavior employs that PUBNC1has zero contents of the Delite HPS bentonite clay whereas PUBNC2has 2% bentonite clay therefore the PUBNC1 has showed some pro-nounced biocompatible behavior than the PUBNC2. While the otherthree samples designated as PUBNC3, PUBNC4 and PUBNC5 havinghigher contents of bentonite nanoclay respectively in their formu-lation showed some toxic and incompatible behavior. Live/deadstaining of cells incubated with the polymer degradation productswith increased bentonite nanoclay contents is not comparable tothe control, thereby suggesting that the higher contents of the clay

have highest cytotoxicity. It can be concluded that for the purposeto modify the mechanical properties with biocompatibility issue,very small amount of bentonite nanoclay is recommended other-wise higher amount have shown some toxic behavior. It is evident

1134 K.M. Zia et al. / International Journal of Biological Macromolecules 49 (2011) 1131– 1136

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ig. 3. Photographs of L-929 cells interaction with sample films of chitin based PUBNC2: 1.0% nanoclay, (c) PUBNC3: 2.0% nanoclay, (d) PUBNC4: 4.0% nanoclay, an

rom Fig. 3 that the cells are small in number and the entire cellsre in round shape in these three samples.

Cell attachment and spreading are examined after staining thebroblast cells cultured on the surface of PUBNC1–5 samples. Thehotographs of Fig. 4 show the attachment and growth of theseells is much higher on the PU film containing zero contents ofhe bentonite clay. The staining result also showed that increasedoncentration of bentonite nanoclay has adverse effect on the bio-ompatibility of the samples. These results are in accorded withhe study that catalyst with organo-metallic character are highlyoxic and leaves an objectionable odor [25] which make the poly-

er film non-biocompatible. In the samples (PUBNC2–5) most ofhe cells are in non-active adhesion and cells are in unlabyrinthine

nd non flattening state. Cell attachment on the sample PUBNC1s higher than all other samples and most of the cells are in activedhesion and flattened on the surface with more spindle-shapedorphology.

-nanocomposites varying nano-clay contents, (a) PUBNC1: without nanoclay, (b)UBNC5: 8.0% nanoclay.

Actin staining of attached L-929 fibroblast cell, as shown inFig. 4, demonstrates dissimilar cell adhesion on the PU bio-nanocomposites surface relative to that on the polystyrene controlsurface. The cells in the sample having zero contents of bentoniteclay contents are attached to the polymer surface and are arrangedin a pattern along the lines of the polymer grooves. The flat fibrob-lastic morphology shows that the cells are anchored well to thesurface of the polymer.

The data in Figs. 3 and 4 imply that the PU bio-nanocompositesand their degradation products have much higher cytotoxic-ity comparable to the control values in most cases. These dataare comparable with those from previous studies indicatingthat organo-metallic character is highly toxic and leaves an

objectionable odor [25] which make the polymer film non biocom-patible. Furthermore, there appear to be comparable differencesin cytotoxicity of degradation products from PUBNC2–5 PU bio-nanocomposites synthesized.

K.M. Zia et al. / International Journal of Biological Macromolecules 49 (2011) 1131– 1136 1135

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ig. 4. Photographs of cellular response of L-929 cells on the surface of films, afteUBNC3: 2.0% nanoclay, (d) PUBNC4: 4.0% nanoclay, and (e) PUBNC5: 8.0% nanocla

.3. Mechanical properties

Measured mechanical parameters are summarized in Table 2hich shows the ultimate tensile strength variations for vari-

us chitin based polyurethane bio-nanocomposites as a functionf Delite HPS® bentonite clay contents in the final solid films.able 2 also demonstrates the ultimate tensile strength of chitinased polyurethane itself without any addition of nano-particles19]. It is clear that the ultimate tensile strength of chitin-basedolyurethane bio-nanocomposites increase with increasing Delite

PS® bentonite clay contents. The prepared bio-nanocomposites

how a plateau behavior up to 4% bentonite nanoclay contentPUBNC4) giving a constant ultimate tensile strength of approx-mately 35 MPa. However, there is tremendous decrease in the

ning with Giemsa, (a) PUBNC1: without nanoclay, (b) PUBNC2: 1.0% nanoclay, (c)

tensile strength using 8% nanoclay contents (PUBNC5). This behav-ior may be due to the incompatibility of the higher contents ofnanoclay (i.e., 8%). Similarly Table 2 illustrates the elongation-at-break for all the samples having 0%, 1%, 2%, 4% and 8% bentonitenanoclay contents. It can clearly be observed that there is a lin-ear reduction of elongation-at-break with increasing Delite HPS®

bentonite clay contents. This could be attributed to the largeraggregations of Delite HPS® bentonite clay contents, as well asthe formation of more aggregated centers with increased DeliteHPS® bentonite clay contents. These increased aggregated cen-

ters would probably act as additional stress centers leading toincreased chances of failure to occur with increased Delite HPS®

bentonite clay contents. The higher values in elongation at breakcould undoubtedly be attributed to the surface characteristics of the

1136 K.M. Zia et al. / International Journal of Biological Macromolecules 49 (2011) 1131– 1136

Table 2Mechanical properties data of polyurethane bio-nanocomposites samples.

Sr. no. Sample code Tensile strength (MPa) Elongation at break (%) Hardness (shore A)

1 PUBNC1a 11.60b 777.42b 85.60b

2 PUBNC2 19.25 709 87.903 PUBNC3 27.80 641 88.984 PUBNC4 35.35 573 90.125 PUBNC5 28.50 438 93.45

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a Polyurethane bio-nanocomposites.b Results from previous study [5].

ower Delite HPS® bentonite clay particles causing a better disper-ion of these particles in the matrix ultimately strengthening thenterfacial interactions between the dispersed phase and the matrixhase. These increased interfacial interactions can maintain or even

mprove ductility due to the more uniformly distributed disper-ions, hence, inhibiting the formation of stress centers essential forotential failure.

. Conclusion

Chitin based polyurethane bio-nanocomposites (PUBNC) wererepared, characterized and evaluated. Molecular characteri-ation was done using FT-IR techniques. Cytotoxicity of theynthesized PU bio-nanocomposite was affected by varying theelite HPS® bentonite nanoclay contents in the chemical com-osition of the final synthesized materials. It is revealed thathe final polymers having 2% Delite HPS® bentonite nanoclayontents is preferred candidates for surgical threads with onoing investigations into their in vitro biocompatibility andon-toxicity. Considered (live/dead) L/D staining data implyhat the degradation products from the PU bio-nanocompositesave increased level of cytotoxicity with increase in ben-onite nanoclay contents. There is tremendous increase in the

echanical properties with increase in contents on the DelitePS® bentonite nanoclay. In this case, the addition of ben-

onite nanoclay contents improved the tensile strength by abouthree times and linear reduction in elongation at break takeslace.

cknowledgments

Financial support of Higher Education Commission (HEC),akistan is highly acknowledged and appreciated in the conductf this work. The extended help of the faculty members of the GCniversity, Faisalabad is also acknowledged.

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