crosslinked silk2

8/4/2019 crosslinked silk2

1/13

Macromolecular Nanotechnology

Preparation and characterization of chitinwhisker-reinforced silk fibroin nanocomposite sponges

Panya Wongpanit a, Neeracha Sanchavanakit b, Prasit Pavasant b,Tanom Bunaprasert c, Yasuhiko Tabata d, Ratana Rujiravanit a,*

a The Petroleum and Petrochemical College, Chulalongkorn University, Bangkok 10330, Thailandb Department of Anatomy, Faculty of Dentistry, Chulalongkorn University, Bangkok 10330, Thailand

c Department of Otolaryngology, Faculty of Medicine, Chulalongkorn University, Bangkok 10330, Thailand

d Department of Biomaterials, Field of Tissue Engineering, Institute for Frontier Medical Sciences, Kyoto University, Kyoto 606-8507, Japan

Received 27 April 2007; received in revised form 27 June 2007; accepted 8 July 2007Available online 19 July 2007

Abstract

For highly porous form such as sponges or scaffolds, the induction of the b-sheet formation of silk fibroin to make thewater-stable materials usually results in their high shrinkage leading to a difficulty in controlling shape and size of mate-rials. Thus, the objective of this study was to improve dimensional stability of silk fibroin sponge by incorporating chitinwhiskers as nanofiller. Chitin whiskers exhibited the average length and width of 427 and 43 nm, respectively. Nanocom-posite sponges at chitin whiskers to silk fibroin weight ratio (C/S ratio) of 0, 1/8, 2/8, or 4/8 were prepared by using afreeze-drying technique. The dispersion of chitin whiskers embedded in the silk fibroin matrix was found to be homoge-neous. The presence of chitin whiskers embedded into silk fibroin sponge not only improved its dimensional stability butalso enhanced its compression strength. Regardless of the chitin whisker content, SEM micrographs showed that all sam-ples possessed an interconnected pore network with an average pore size of 150 lm. To investigate the feasibility of thenanocomposites for tissue engineering applications, L929 cells were seeded onto their surfaces, the results indicated thatsilk fibroin sponges both with and without chitin whiskers were cytocompatible. Moreover, when compared to the neatsilk fibroin sponge, the incorporation of chitin whiskers into the silk fibroin matrix was found to promote cell spreading. 2007 Elsevier Ltd. All rights reserved.

Keywords: Silk fibroin; Chitin whisker; Nanocomposite; Scaffold

1. Introduction

Silk fibers from the silkworm Bombyx mori havebeen used commercially as biomedical sutures for

decades but some biocompatibility problems havebeen reported during the past 20 years [13]. How-ever, after silk sericin is properly extracted, silkfibers and regenerated silk fibroin films exhibit com-parable biocompatibility in vitro and in vivo withother commonly used biomaterials such as polylac-tic acid and collagen [1,4]. Regenerated silk fibroinis also biodegradable [57], and a number of cells,

0014-3057/$ - see front matter 2007 Elsevier Ltd. All rights reserved.

doi:10.1016/j.eurpolymj.2007.07.004

* Corresponding author. Tel.: +66 2218 4132; fax: +66 22154459.

E-mail address: [email protected] (R. Rujiravanit).

European Polymer Journal 43 (2007) 41234135

www.elsevier.com/locate/europolj

EUROPEAN

POLYMER

JOURNAL

MACROMOLEC

ULARNANOTECHNOL

OGY
mailto:[email protected]:[email protected]


2/13

e.g. L929 cells [8], endothelial cells [9], keratino-cytes, osteoblasts [10], fibroblasts [10,11], bone mar-row stromal cells [12], and bone marrow-derivedmesenchymal stem cells [13], have been shown togrow well on its surface. Because of its high cyto-

compatibility, the regenerated silk fibroin is aninteresting scaffolding biomaterial applicable for awide range of target tissues.

Before actual use, the regenerated silk fibroinrequires post-treatment in order to induce the con-formation transition from random coil to b-sheetstructure to attain a water-stable material. Post-treatments of silk fibroin can be done by using anaqueous alcohol solution [14], thermal annealing[15] or physical stretching [7]. Among them, themethod of annealing with solvents is much morewidely used than others because it is simple and per-

formed under mild condition; however, this transi-tion induction causes the shrinkage of the materialleading to its poor dimensional stability, especiallyfor highly porous material. In this study, silk fibroinsponges also show a relatively low strength. Nor-mally, materials must have sufficient mechanicalintegrity to resist handling during implantationand in vivo loading. Therefore, these two importantlimitations of silk fibroin need to be solved.

In the plastic industry, the use of inorganic fillershas been a common way to improve mold shrink-age, thermal and mechanical properties of thermo-plastics. The effect of filler on the mechanical andother properties of the composites depend greatlyon its shape, particle size, aggregated size, surfacecharacteristics, and degree of dispersion. Generally,the mechanical properties of the composites filledwith micron-sized filler particles are inferior to thosefilled with nanoparticles of the same filler [16,17].Intuitively, the adaptation of knowledge in thermo-plastic nanocomposite is expected to be useful forbiomaterials.

It is known that the mechanical properties of the

composites are strongly related to the aspect ratio ofthe filler particles. Based on this, the chitin whiskers,having a wide range of aspect ratios, i.e. 10120,depending on types of sources, have been exten-sively studied as reinforcing nanofillers in recentyears [1823]. Chitin or poly(b(1-4)-N-acetyl-D-glu-cosamine) is one of the most abundant polysaccha-rides found in nature. It can be found in the skeletalmaterials of crustaceans, cuticles of insects, and cellwalls of various fungi. Chitin is known to be cyto-compatible [2426] and biodegradable [27]. In addi-tion, chitin whiskers have been successfully

prepared from crab shells [1921,28,29], squid pens[30], tubes of Riftia pachyptila tubeworms [18], andshrimp shells [22,23].

In this study, chitin whisker-reinforced silk fibroinnanocomposite sponges were fabricated using a

freeze-drying technique. The presence of chitin whis-ker embedded in silk fibroin matrix was expected toimprove their dimensional stability as well as themechanical properties. The effect of the whisker con-tent on the morphology of the sponges, and the inter-action between chitin whiskers and silk fibroin werealso discussed. The dispersion of chitin whiskers infibroin matrix was investigated. In addition, the mor-phologies of L929 cells on silk fibroin sponges withand without chitin whiskers were observed.

2. Experimental

2.1. Materials

Raw silk fiber, Bombyx Mori, was obtained fromQueen Sirikit Sericulture Center, Saraburi Province(Thailand). Shell ofPenaeus merguiensis shrimp waskindly supplied by Surapon Foods Public Co., Ltd(Thailand). All of the materials (analytical grade)were used as received.

2.2. Preparation of chitin whisker suspension

After decalcification and deproteinization, thedegree of deacetylation (DD) of the obtained chitinwas determined by Fourier-transformed infraredspectroscopy (FT-IR) following the method of Bax-ter et al. [31] and was found to be 20%.

The preparation of chitin whiskers was modifiedfrom the method of Morin & Dufresne [18]. Briefly,chitin whisker suspension was prepared by hydro-lyzing chitin flake (


3/13

2.3. Preparation of aqueous silk fibroin solution

The raw silk fibers of Bombyx mori were boiledfor 15 min in an aqueous solution of 0.05%Na2CO3. The boiling process was repeated two

times to remove sericin. The fibers were then rinsedthoroughly with hot water and dried at 40 oC over-night. After drying, the degummed silk fibers weredissolved in a solvent mixture of CaCl2:ethanol:H2O(molar ratio = 1:2:8) at 78 oC. The undissolved par-ticle was filtered out using filter cloth. The filtratedsolution was subsequently dialyzed in distilled waterfor 4 days by changing the media daily, followed bycentrifugation at 10,000 rpm for 10 min. The as-pre-pared aqueous silk fibroin concentration was6.32 wt%.

2.4. Preparation of chitin whisker-reinforced silk fibroin sponges

The as-prepared chitin whisker suspension wereadded to distilled water to achieve a chitin whiskerconcentration at 0, 0.125, 0.25 or 0.50 wt% (a con-centration based on the final volume including dis-tilled water, chitin whisker suspension, and silkfibroin solution) with a volume of 8.42 ml. The sus-pensions were then treated by ultrasonication for10 min. After the suspension was kept at 4 oC for10 min, 1.58 ml of 6.32 wt% aqueous silk fibroinsolution was added to the suspension with slowmechanical stirring. The obtained chitin whisker/silk fibroin suspension was equivalent to chitinwhiskers to silk fibroin weight ratio (C/S ratio) of0, 1/8, 2/8, or 4/8. The mixture suspension was fur-ther stirred mechanically for 10 min before beingpoured with 1.6 ml into each well of a 24 multi-wellsculture plate (COSTAR, Corning Inc., NY, USA).The suspension was frozen at 40 oC overnightunder controlled cooling rate, followed by freeze-drying under vacuum condition of less than 10 Pa

for 24 h to obtain the nanocomposite sponges.

2.5. Methanol treatment

The silk fibroin sponges both with and withoutchitin whiskers were immersed in an aqueous meth-anol solution at a concentration of 90% (v/v) for10 min. After excessively washing with distilledwater, the methanol-treated sponges were subse-quently frozen at 40 oC overnight under con-trolled cooling rate, followed by freeze-dryingunder vacuum condition of less than 10 Pa for 24 h.

2.6. Characterization of chitin whisker-reinforced silk

fibroin samples

The volume of the sponges before and aftermethanol treatment was determined in order to cal-

culate the percent shrinkage (i.e. shrinkage (%) =(ViVf)/Vi 100; where Vi and Vf represent the vol-ume of the sponges before and after methanol treat-ment, respectively). The sample number was five foreach experimental group.

The compression test was performed for thedried sponges of the methanol-treated samples atroom temperature on the Texture Analyzer(TA.XT2i, England) at a crosshead speed of0.5 mm/min. The compressive modulus was calcu-lated from the initial slope of the linear portion ofthe stressstrain curve [32]. In order to eliminate

the influence of additional weight of the spongesfrom mixing with chitin whiskers and the volumechange after methanol treatment, the compressivemodulus was then divided by the bulk density,namely the specific modulus [33]. The experimentwas performed for four sponges per sample.

FT-IR spectrophotometry was used to verify thechemical structure and conformation of the as-pre-pared silk fibroin sponge, methanol-treated spongesat various C/S ratios, and chitin whisker film. Themeasurements were carried out on a Thermo Nico-let Nexus 671 FT-IR (32 scans at a resolution of4 cm1). A MettlerToledo differential scanning cal-orimetry (DSC) model 822e/400 was used to evalu-ate thermal properties of the methanol-treated silkfibroin sponges with and without chitin whiskers,and the freeze-dried chitin whiskers (about 3 mg)under N2 atmosphere at a heating rate of10 K min1 from 50 to 450 oC.

The inner microstructure of the as-prepared silkfibroin sponges with and without chitin whiskers,and the corresponding methanol-treated spongeswas observed by a scanning electron microscope

(SEM, JSM-5410LV, Jeol, Japan) at a voltage of15 kV. The sponge was cut with a razor blade.The cross-section of the sponges was coated withgold on the ion sputter (SCD040, Lichtenstein) at50 mTorr and 15 mA for 4 min.

The morphological appearance and sizes of theas-prepared chitin whiskers were observed using aJEOL JEM-1230 transmission electron microscope(TEM). Samples were prepared from a drop of adilute chitin whisker suspension which was depos-ited and left to dry on a carbon coated copper grid.The dispersion of chitin whiskers in the silk fibroin

P. Wongpanit et al. / European Polymer Journal 43 (2007) 41234135 4125

MACROMOLEC

ULARNANOTECHNOL

OGY


4/13

matrix was also observed with TEM by embeddingthe sponge at C/S ratio of 4/8 with Aradite resin.The sample was then cut to ultrathin sections(200 nm) and placed on a copper grid with a carboncoating. The observations were carried out at an

accelerating voltage of 120 kV.The universal hardness (H) of silk fibroin and

chitin whisker/silk fibroin films at C/S ratio of 4/8was carried out using Nanoscope (R) IV scanningprobe microscope (Veeco/Digital Instruments, CA)in a nano-indentation mode in order to investigatethe influence of hardness on cell spreading. The dia-mond-tipped probe made a prescan to measure thesurface profile. The probe then applied the forceat a trigger threshold of 3 V (230 lN) onto thescanned film surfaces (spring constant of 291 N/mspecified by the manufacturer). The hardness were

calculated from H = Force/A, where A is the pro-jected area at peak load.

2.7. Cells and cell culture

A cell line of L929 (ECACC Cat. No. 85011425)mouse connective tissue, which is a fibroblast-likecell, was cultured in Dulbeccos modified eaglesmedium (DMEM; Sigma-Aldrich, USA) supple-mented with 10% fetal bovine serum (FBS,BIOCHROM AG), together with 100 units/ml pen-icillin (GIBCO) and 100 lg/ml streptomycin(GIBCO) at 37 C in a wet atmosphere containing5% CO2. When the cells reached 80% confluence,they were trypsinized with 0.25% trypsin containing1 mM EDTA (GIBCO) and counted by a hemacy-tometer (Hausser Scientific, USA) before use inthe experiments.

2.8. Cell spreading analysis

In these experiments, each prepared sponge wasplaced in a 24-well culture plate and sterilized by

70% ethanol solution for 30 min. Thereafter, itwas washed twice with phosphate buffer saline(PBS, pH 7.4) and subsequently washed with culturemedia. Before cell seeding, 500 ll of culture mediumwas pipetted into each well of the 24-well plate.1 105 of L929 cells were seeded into each well ofthe culture plate and allowed to attach to the meth-anol-treated silk fibroin and chitin whisker/silkfibroin sponges (at C/S ratio of 4/8) for 6 h or24 h. After each sample was rinsed twice withPBS, they were fixed with 3% glutaraldehyde for30 min, followed by two rinses with a 0.2 M phos-

phate buffer. Then the samples were dehydratedthrough a series of graded ethanol and subsequentlydried in a critical point dryer with liquid CO2. Thesamples were gold sputtered in a vacuum at50 mTorr and 15 mA for 4 min.

Cell spreading was evidenced from SEM micro-graphs taken by a JSM-5410LV scanning electronmicroscope at a voltage of 15 kV. The cells thatadopted a flattened, polygonal shape, with filopo-dia- and lamellipodia-like extensions were regardedas spreading cells. In contrast, the cells that resistedto washing and remained tethered to the surfacewere regarded as non-spreading cells. The percentcell spreading was quantified by dividing the num-ber of spread cells by the total number of boundcells (N= 100 cells) [34]. Standard deviations werecalculated from two independent experiments.

2.9. Statistical analysis

All the results were statistically analyzed by theunpaired Students t-test and the result withP< 0.05 was considered to be statistically signifi-cant. Data were expressed as the mean the stan-dard deviation of the mean (SD).

3. Results and discussion

3.1. Morphology and size of chitin whisker

As stated, several investigations indicated thatthe regenerated silk fibroin exhibited a number ofprerequisites for tissue engineering scaffolds; how-ever, the shrinkage of the regenerated silk fibroinafter methanol treatment was scarcely concerned.Although there were some researchers whoattempted to fabricate silk fibroin without methanoltreatment, the proposed methods were restricted tothe preparation of only the film using a very highconcentration of silk fibroin (>8 wt%) [7,35]. How-

ever, a very interesting method to prepare an insol-uble silk fibroin sponge without methanol treatmentwas proposed by the work of Kim et al. [36] whoprepared the silk fibroin sponges via a salt leachingtechnique.

For commercially available products, type I Col-lagen-Glycosaminoglycan (CG) sponges are suc-cessfully being used to regenerate skin in burnpatients; this material received FDA approval forclinical use in 1996. Porous CG sponges have beenprimarily fabricated using a freeze-drying technique[37]. The structure of the sponge is controlled by the

4126 P. Wongpanit et al. / European Polymer Journal 43 (2007) 41234135

MACROMOLEC

ULARNANOTECHNOL

OGY


5/13

final temperature of freezing and the heat transferprocesses associated with freezing. Furthermore,the formation of ice crystals within the suspensionis influenced by both the rate of nucleation of icecrystals and the rate of heat and protein diffusion

[38]. In this study, the chitin whisker-reinforced silkfibroin nanocomposite sponges were successfullyprepared via a freeze-drying technique because chi-tin whisker suspension displayed the colloidalbehavior and there was no precipitation of chitinwhiskers occurred during mixing with the silkfibroin solution.

Considering morphology and size of chitin whis-ker, Fig. 1a is the TEM micrograph of a dilute sus-pension of chitin whiskers from the acid hydrolysisof shrimp chitin. It was found that the suspensionwas composed of chitin fragments containing both

individual microcrystals and aggregated microcrys-tals. The chitin fragments were consisted of slenderrods with sharp points which had a broad distribu-tion in size. The length of the chitin fragments ran-ged from 180 to 820 nm, while the width rangedfrom 8 to 74 nm. The average length (L) and width(d) of these whiskers were about 427 and 43 nm,respectively, and the average aspect ratio (L/d)was of 10. These dimensions are in the range ofthe previously reported values for chitin whiskersobtained from the same source of chitin (L = 150to 800 nm and d = 570 nm [22,23]). However, thedimensions of chitin whiskers prepared from othersources having a wide range reported values suchas crab shells (L = 50300 nm and d = 68 nm[28]; L = 100600 nm and d = 440 nm [1921];and L = 100650 nm and d = 1080 nm [29]) squidpens (L = 50300 nm and d = $10 nm [30]), orRiftia tubes (L = 500 nm to 10 lm and d =$18 nm [18]).

In addition, the chitin suspension exhibited col-loidal behavior due to the presence of the positivecharge on chitin whiskers. The shrimp chitin used

to prepare chitin whiskers in this work had a degreeof deacetylation of 20%. It implied that there weresome amino groups over the surface of chitin whis-ker. At neutral pH, the number of protonatedamino groups (NH3

+) on the surface of chitinwhiskers was slightly lower than that of aminogroups (NH2) because pH of the suspension washigher than pKa of NH2 ($6.4) [39]. However,the presence of the positive charge of the protonatedamino groups on the whisker surface was sufficientenough to stabilize the colloidal behavior of the chi-tin suspension [40].

3.2. Dimensional stability

In order to determine dimensional stability aftermethanol treatment, the changes in the volume ofthe silk fibroin sponges both before and after metha-nol treatment was measured. Fig. 2 illustrates thepercent shrinkage of silk fibroin sponges both withand without chitin whiskers. It was found that thepercent shrinkage of the silk fibroin sponges couldbe significantly reduced at all C/S ratios. In addition,the percent shrinkage decreased with an increasingof the whisker content and finally reached the

Fig. 1. TEM micrographs of chitin whisker; Bar, 2 lm (a), andTEM micrographs of ultrathin section of chitin whisker/silkfibroin sponge at C/S ratios of 4/8; Bar, 200 nm (b).


MACROMOLEC

ULARNANOTECHNOL

OGY


6/13

plateau at C/S ratio of 2/8. Interestingly, at only C/Sratio of 1/8, the percent shrinkage of the spongesreduced appreciably from 35.7 to 16.0.

It is known that methanol treatment of silkfibroin induces the transition of random coil confor-mation to b-sheet structure which results in theshrinkage of materials. After incorporating chitinwhiskers into silk fibroin sponge, the shrinkage ofthe nanocomposite sponges was appreciablyreduced. To explain the shrinkage reduction of thenanocomposite sponges, conformation of silkfibroin existent in the methanol-treated nanocom-posite sponges had to be clarified whether the pres-ence of chitin whiskers inhibited or disrupted b-sheet formation of silk fibroin. Fig. 3 illustrates theFT-IR spectra of the as-prepared silk fibroin sponge,the methanol-treated sponges with various C/Sratios, and the chitin whisker film. The neat silkfibroin sponge before methanol treatment exhibitedthe characteristic absorption peaks which wereassigned to the random coil conformation at 1648(amide I), 1536 (amide II) and 1237 cm1 (amide

III) [41]. After the silk fibroin sponge was treatedwith methanol, the peaks of amide I and amide IIwere shifted to 1624 and 1516 cm1, respectively,and a new absorption shoulder was observed at1697 and 1268 cm1, which are the characteristicabsorptions ofb-sheet form of silk fibroin [42]. Forchitin whiskers, the characteristic absorption peaksof chitin whiskers were presented at 1658, 1116 and1075 cm1, which were assigned to amide I, second-ary alcohol, and primary alcohol, respectively [43].

Considering the FT-IR spectra of the nanocom-posite, it is obvious that the sponges at all C/S ratios

showed the characteristic absorption peaks of b-sheet structure at 1624, 1516 cm1, and the absorp-tion shoulder at 1697 and 1268 cm1. In addition,the absorption peak of primary alcohol of chitin

whiskers at 1075 cm1

also present and the intensityof this peak increased with an increasing of C/Sratio. Thus the presence of chitin whiskers in silkfibroin sponge did not inhibit the b-sheet formationof silk fibroin after methanol treatment. This indi-cated that the reduction of the shrinkage of thenanocomposite sponges was not resulted from theinhibition of the b-sheet formation. Dissolution testof the sponges also confirmed the FT-IR results, theas-prepared nanocomposite sponges could be dis-solved within a few minutes after submerging in dis-tilled water for all C/S ratios. In contrast, the

Fig. 2. Percent shrinkages of chitin whisker/silk fibroin spongesat various C/S ratio. *, P< 0.05; significant against the percentshrinkage of the neat silk fibroin sponges.

Fig. 3. FT-IR spectra of the as-prepared silk fibroin sponge (A),methanol-treated sponges at various C/S ratios (B to E), andchitin whisker film (F). Arrows indicate the absorption shoulder.


MACROMOLEC

ULARNANOTECHNOL

OGY


7/13

methanol-treated nanocomposite sponges were sta-ble in water regardless of C/S ratios.

Chen et al. [44] suggested that the low shrinkageduring the curing of the epoxy resin-based nano-composite was caused by the strong interfacial inter-

actions between the resin and nanosilica fillers. Inorder to investigate the interaction between silkfibroin and chitin whiskers, the thermal propertiesof silk fibroin and their nanocomposite samplesmight provide useful information. Fig. 4 shows theDSC thermograms of the methanol-treated chitinwhisker/silk fibroin sponges at different C/S ratios,

and the freeze-dried chitin whiskers within the tem-perature range of 50450 oC. Apparently, two differ-ent types of thermal transition were observed inthese thermograms. It was postulated that the wideendothermic peak at temperature below 190 oC was

a result of the loss of moisture within the samples,while another peak at higher temperature regionwas caused by the thermal decomposition of thesamples.

According to Fig. 4A, the decomposition temper-ature of neat silk fibroin appeared to be at thebroader endothermic peak of 287 oC and at thesharper endothermic peak of 304 oC. The presenceof the whiskers at C/S ratio of 1/8 (see Fig. 4B)induced the lowering of the sharp peak of thedecomposition temperature from 304 to 298 oC withthe change in thermal transition from the rough to

smooth curve but the wide endothermic peak ofthe decomposition temperature (288 oC) was stillsimilar. The further increase in the amount of chitinwhiskers (see Fig. 4C and D) resulted in the absenceof the sharp endothermic peak of the decompositiontemperature. Analogous to C/S ratio of 1:8, thebroad endothermic peak of the decomposition tem-perature (287 oC) was still unchanged. By carefulconsideration of the thermograms present inFig. 4C and D (belonging to the nanocompositewith C/S ratio of 2/8 and 4/8), there was a new exo-thermal curve, which could not be observed in thethermograms of both neat silk fibroin and chitinwhiskers, due to the shift of the baselines in therange of 385415 oC. It should be noted that thedecomposition of the chitin whiskers caused thecurve shifting (exothermic) of the baseline in thetemperature range of 260410 oC (see Fig. 4E).

Compared to the neat silk fibroin and chitinwhiskers, the noticeable change in the thermaldecomposition behavior of the nanocompositesmight imply to the interfacial interactions (mainlyhydrogen bonding) forming between polar groups

present in the chemical structures of silk fibroinand chitin whiskers in the nanocomposite sponges.These interactions restricted the mobility of silkfibroin chains and also reduced inter- and intra-molecular interactions between silk fibroin chains.Therefore, the shrinkage of the silk fibroin nano-composite sponges after methanol treatment couldbe reduced.

In addition, a short single crystal fiber, i.e. chitinwhisker, is a highly crystalline material so its vol-ume is still unchanged after methanol treatment.Hence, the incorporation of dimension-stable fillers

Fig. 4. DSC thermograms of the methanol-treated silk fibroinsponge (A), the methanol-treated chitin whisker/silk fibroinsponges having different C/S ratios, 1/8 (B), 2/8 (C), 4/8 (D),and the freeze-dried chitin whisker (E).


MACROMOLEC

ULARNANOTECHNOL

OGY


8/13

into the matrix resulted in a reduction in the percentshrinkage of the sponges, which also decreased withan increasing amount of the stable filler. Theseresults are analogous to the study of Hokugoet al. [32] who demonstrated that the incorporation

of poly(glycolic acid) micro-fiber (whose volumewas not changed during submersion in the culturemedia) into fibrin sponges resulted in the reductionof the percent shrinkage compared to the neat fibrinsponge, and the percent shrinkage of the spongealso decreased with increasing of the fiber content.

3.3. Compression test

Typically, the purposes or advantages of theincorporation of the nanofillers into the matrix areto mimic the target organ component [45,46], to

enhance the biological responses, i.e. cell adhesion,proliferation [46] or biodegradation rate [47], and/or frequently to promote the mechanical properties[45,47,48].

The specific moduli of the methanol-treated silkfibroin sponges with and without chitin whiskersare shown in Table 1. The specific modulus was cal-culated by dividing the compressive modulus by thebulk density. Apparently, although the specific mod-ulus of the methanol-treated nanocomposite at C/Sratio of 1/8 was comparable to that of the neat silkfibroin sponges, the specific moduli of those at C/Sratio of 2/8 and 4/8 were statistically significanthigher by 2.6 and 7.5 orders of magnitude, respec-tively, when compare to that of the control sample.Favier et al. [49,50] found that when cellulose whis-ker content was reached in percolation threshold,the mechanical properties of the cellulose whisker-reinforced polymer nanocomposite films were sub-stantially enhanced. They demonstrated that thesubstantial enhancement of the mechanical proper-ties was caused by the hydrogen-bonding between

the whiskers that hold the percolating network ofthe whiskers. They further suggested that good dis-persion of the whiskers was also another importantfactor that promoted the mechanical properties.

Fig. 1b is the TEM micrograph which illustrates

the homogeneous dispersion of chitin whiskersembedded in the silk fibroin matrix. The morphol-ogy of chitin whiskers exhibited the individualmicrocrystals without aggregation. Embedded chi-tin whiskers displayed the width in the range of 768 nm, while its average was 21 nm which correlatedto the size of bare chitin whiskers in Section 3.1 eventhe average was slightly lower. Therefore, whenincorporating chitin whiskers into silk fibroin at asuitable ratio, the compressive properties of thesponges were substantially improved. Note thatthe TEM micrograph displays only the perpendicu-

lar and tilt cross-section of the whiskers withoutshowing the entire rod of the whisker because theTEM micrograph was taken from the ultrathin sam-ple with the thickness of 200 nm.

3.4. Morphological appearance of nanocomposite

sponges

For tissue engineering applications, the cellularstructure of the sponge is one of the most importantfactors determining whether regeneration of a newtissue is successful or not. In general, the cellularstructure of the sponge must be designed to satisfya number of requirements, e.g. the pore size mustbe in a critical range (usually 100200 lm) and theporosity must be interconnected to allow theingrowth of cells, vascularization and the diffusionof nutrients [37].

Fig. 5 is the SEM micrographs of silk fibroinsponges with and without chitin whiskers afterimmersing in a 90% (v/v) aqueous methanol solutionfor 10 min in a comparison with the as-preparedsponge. Regardless of chitin whisker content, an

interconnected pore network was observed with anaverage pore size of 150 lm for the sponges bothbefore (see Fig. 5a, b, c and d) and after methanoltreatment (see Fig. 5e, f, g and h), despite the factthat a contraction occurred in all methanol-treatedsamples especially a substantial contraction of themethanol-treated silk fibroin sponges (viz. the per-cent shrinkage of the sponges was 35.7, see Fig. 2).In a separated experiment, all of the dried metha-nol-treated samples could absorb and sink indistilled water within a few seconds after dropping.This observation confirmed the presence of an

Table 1Bulk densities and specific moduli of the methanol-treated chitinwhisker/silk fibroin sponges at a various C/S ratios

Methanol-treatedsponges at differentC/S ratios

Bulk density(g cm3) 102

Specific modulus(kPa g1 cm3) 102

0 1.595 0.071 4.435 0.641:8 1.475 0.023 5.005 1.322:8 1.545 0.042 11.85 2.40*

4:8 1.885 0.051 33.35 5.21*

* =P


9/13

Fig. 5. Cross-sectional SEM micrographs of chitin whisker/silk fibroin sponges at C/S ratio of 0 (a), 1/8 (b), 2/8 (c) or 4/8 (d); And thecorresponding methanol-treated sponges at C/S ratio of 0 (e), 1/8 (f), 2/8 (g) or 4/8 (h).


MACROMOLEC

ULARNANOTECHNOL

OGY


10/13

interconnected pore network in the materials, corre-sponding to the SEM observation.

3.5. Morphological appearance of cell adhesion and

analysis of cell spreading

Undoubtedly, both neat silk fibroin and chitinare not cytotoxic. However, the morphology ofthe attached cells on the sponges had to be observedin order to confirm the feasibility of the chitin whis-ker-reinforced silk fibroin nanocomposite spongesfor tissue engineering applications.

Fig. 6 shows the SEM micrographs of L929 cellscultured on the methanol-treated silk fibroin andchitin whisker/silk fibroin sponges at C/S ratio of4/8. SEM micrographs revealed the cell morphologyand interaction between cells and sponges. At 6 h of

cultivation, it was observed that the cells could

attach on both types of sponges with their filopodia.The cells also exhibited different shapes, includingflattening, typical elongated fibroblast cell shapes,and spherical shapes on both types of sponge (seeFig. 6a and c). At 24 h of cultivation (see Fig. 6b

and d), the number of cells with extended filopodiawas observed to be relatively higher (compared to at6 h) and they showed a typical cell morphology onboth types of sponges.

Interestingly, an observed number of flattenedcells on the methanol-treated chitin whisker/silkfibroin sponges were higher than that on the metha-nol-treated neat silk fibroin sponges. At this point, itwas interested to quantify the percent cell spreadingon both types of matrix to determine the influence ofthe incorporated chitin whiskers on cell spreading.Cell spreading analysis revealed that there was no

statistically significant difference between both types

Fig. 6. SEM micrographs of attached cells on methanol-treated silk fibroin sponges for (a) 6 and (b) 24 h of cultivation and SEMmicrographs of attached cells on methanol-treated chitin whisker/silk fibroin sponges at C/S ratio of 4/8 for (c) 6 and (d) 24 h ofcultivation.


MACROMOLEC

ULARNANOTECHNOL

OGY


11/13

of the substrates at 6 h of cultivation; however, thecells could attach to the chitin whisker/silk fibroinsponges with significantly higher percent cell spread-ing compared to the neat silk fibroin sponges at 24 hof cultivation (see Fig. 7). It should be noted that thepercent cell spreading at 24 h of cultivation on chitinwhisker/silk fibroin sponges was almost twice (61%)compared to that on the neat silk fibroin sponges(31%).

From literature, a number of physicochemicalsurface properties; surface free energy [51,52], sur-face charge [53], surface chemistry [54], and surfacetopography [55], have been shown to affect cellspreading. Recently, the mechanical properties ofsubstrates have been demonstrated to affect cellspreading as well. Pelham and Wang [56] preparedsubstrates from polyacrylamide sheets with different

crosslinking densities and the surfaces of the pre-pared substrates were coated with type I collagenin order to vary the rigidity while maintaining thechemical environment. They found that cells onflexible substrates showed reduced spreading whencompared to that on rigid substrates. Fereol et al.[57] also found similar results even with substrateshaving different surface chemistries.

Generally, the incorporation of rigid fillers intothe polymeric matrixes could improve their rigidityor hardness. In the present study, the incorporationof chitin whiskers at C/S ratio of 4/8 significantly

enhanced the rigidity of the silk fibroin matrix ascompared between the universal hardness of the silkfibroin films with and without chitin whiskers;1.55 0.21 and 1.055 0.12 GPa, respectively.Therefore, the enhancement of the rigidity of silk

fibroin films by incorporation of the chitin whiskersmight cause better cell spreading.

It could be concluded that both methanol-treatedsilk fibroin and chitin whisker/silk fibroin spongesexhibited the absence of cytotoxicity because thecells attached on the substrates showed the normalcell morphology. In addition, the incorporation ofchitin whiskers into the silk fibroin matrix enabledthe significant promotion of the percent cell spread-ing of the L929 cells.

4. Conclusion

Because of the colloidal behavior of the chitinwhiskers and no observation of a precipitation aftermixing chitin whiskers with silk fibroin, a freeze-drying technique can be used for fabrication of thesponge form and produced the materials having apore size in a critical range with interconnectingpore network. The incorporating chitin whiskersinto the silk fibroin matrix not only promoted thedimensional stability but also enhanced the mechan-ical properties of the silk fibroin sponges. Moreover,the nanocomposite sponges exhibited the absence ofthe cytotoxicity as well as promoting cell spreading.Thus, all of these results might indicate the potentialutility of this nanocomposite system for furtherexploration as a scaffolding material.

Acknowledgements

The authors acknowledge Associate ProfessorPitt Supaphol for their stimulating discussion. Thiswork was supported in part by the National Re-search Council of Thailand (through a research grant

for Multidisciplinary Research Series in TissueEngineering project), Chulalongkorn University(through a research grant from the RatchadaphisekSomphot Endowment Fund), the Thailand ResearchFund (through the Royal Golden Jubilee Ph.DProgram i.e., RGJ grant), the Petroleum and Petro-chemical Technology Consortium (through agovernmental loan from the Asian DevelopmentBank), and the Petroleum and PetrochemicalCollege, Chulalongkorn University. The authorswish to thank Queen Sirikit Sericulture Center (Sara-buri Province, Thailand) and Surapon Foods Public

Fig. 7. Percent cell spreading of the L929 cells on methanol-treated neat silk fibroin sponges and methanol-treated chitinwhisker/silk fibroin sponges having C/S ratio at 4:8, *, P< 0.05;significant against the percent cell spreading of methanol-treatedneat silk fibroin sponges.


MACROMOLEC

ULARNANOTECHNOL

OGY


12/13

Co., Ltd. (Thailand) for supplying essential materialsfor this study.

References

[1] Altman GH, Diaz F, Jakuba C, Calabro T, Horan RL, ChenJ, et al. Silk-based biomaterials. Biomaterials 2003;24:40116.

[2] Wen CM, Ye ST, Zhou LX, Yu Y. Silk-induced asthma inchildren:a reportof 64 cases.Annals Allergy 1990; 65:35875.

[3] Soong HK, Kenyon KR. Adverse reactions to virgin silksutures in cataract surgery. Ophthalmology 1984;91:47983.

[4] Meinel L, Hofmann S, Karageorgiou V, Kirker-Head C,McCool J, Gronowicz G, et al. The inflammatory responsesto silk films in vitro and in vivo. Biomaterials 2005;26:14755.

[5] Li M, Ogiso M, Minoura N. Enzymatic degradationbehavior of porous silk fibroin sheets. Biomaterials 2003;24:35765.

[6] Arai T, Freddi G, Innocenti C, Tsukada M. Biodegradationof Bombyx mori silk fibroin fibers and films. J Appl PolymSci 2004;91:238390.

[7] Jin H-J, Park J, Karageorgiou V, Kim U, Valluzzi R, CebeP, Kaplan DL. Water-stable silk films with reduced beta-Sheet content. Adv Funct Mater 2005;15:12417.

[8] Minoura N, Aiba S, Gotoh Y, Tsukada M, Imai Y.Attachment and growth of cultured fibroblast cells on silkprotein matrices. J Biomed Mater Res 1995;29:121521.

[9] Unger RE, Peters K, Wolf M, Motta A, Migliaresi C,Kirkpatrick CJ. Endothelialization of a non-woven silkfibroin net for use in tissue engineering: growth and generegulation of human endothelial cells. Biomaterials 2004;25:513746.

[10] Unger RE, Wolf M, Peters K, Motta A, Migliaresi C,Kirkpatrick CJ. Growth of human cells on a non-woven silkfibroin net: a potential for use in tissue engineering.Biomaterials 2004;25:106975.

[11] Yamada H, Igarashi Y, Takasu Y, Saito H, Tsubouchi K.Identification of fibroin-derived peptides enhancing theproliferation of cultured human skin fibroblasts. Biomate-rials 2004;25:46772.

[12] Jin H-J, Chen J, Karageorgiou V, Altman GH, Kaplan DL.Human bone marrow stromal cell responses on electrospunsilk fibroin mats. Biomaterials 2004;25:103947.

[13] Li C, Vepari C, Jin H-J, Kim HJ, Kaplan DL. Electrospunsilk-BMP-2 scaffolds for bone tissue engineering. Biomate-rials 2006;27:311524.

[14] Nam J, Park YH. Morphology of regenerated silk fibroin:effects of freezing temperature, alcohol addition, andmolecular weight. J Appl Polym Sci 2001;81:300821.

[15] Saitoh H, Ohshima K, Tsubouchi K, Takasu Y, Yamada H.X-ray structural study of noncrystalline regenerated Bombyxmori silk fibroin. Int J Biol Macromol 2004;34:259365.

[16] Sumita M, Shizuma T, Miyasaka K, Ishikawa K. Effect ofreducible properties of temperature, rate of strain, and fillercontent on the tensile yield stress of nylon 6 composite filledwith ultrafine particles. J Macromol Sci Phys 1983;B22:60118.

[17] Sumita M, Tsukumo T, Miyasaka K, Ishikawa K. Tensileyield stress of polypropylene composites filled with ultrafineparticles. J Mater Sci 1983;18:175864.

[18] Morin A, Dufresne A. Nanocomposites of chitin whiskersfrom Riftia Tubes and poly(caprolactone). Macromolecules2002;35:21909.

[19] Nair KG, Dufresne A. Crab shell chitin whisker reinforcednatural rubber nanocomposites. 1. Processing and swellingbehavior. Biomacromolecules 2003;4:65765.

[20] Nair KG, Dufresne A. Crab shell chitin whisker reinforcednatural rubber nanocomposites. 2. Mechanical behavior.Biomacromolecules 2003;4:66674.

[21] Nair KG, Dufresne A. Crab shell chitin whisker reinforcednatural rubber nanocomposites. 3. Effect of chemical mod-ification of chitin whiskers. Biomacromolecules 2003;4:183542.

[22] Sriupayo J, Supaphol P, Blackwell J, Rujiravanit R.Preparation and characterization of a chitin whisker-rein-forced chitosan nanocomposite films with or without heattreatment. Carbohyd Polym 2005;62:1306.

[23] Sriupayo J, Supaphol P, Blackwell J, Rujiravanit R.Preparation and characterization of alpha-chitin whisker-reinforced poly(vinyl alcohol) nanocomposite films with or

without heat treatment. Polymer 2005;46:563744.[24] Howling GI, Dettmar PW, Goddard PA, Hampson FC,Dormish M, Wood EJ. The effect of chitin and chitosan onthe proliferation of the human skin fibroblasts and kerati-nocytes in vitro. Biomaterials 2001;22:295966.

[25] Chatelet C, Damour O, Domard A. Influence of the degreeof acetylation on some biological properties of chitosanfilms. Biomaterials 2001;22:2618.

[26] Wongpanit P, Sanchavanakit N, Pavasant P, Supaphol P,Tokura S, Rujiravanit R. Preparation and characterizationof microwave-treated carboxymethyl chitin and carboxym-ethyl chitosan films for potential use in wound careapplication. Macromol Biosci 2005;5:100112.

[27] Tomihata K, Ikada Y. In vitro and in vivo degradation of

films of chitin and its deacetylated derivatives. Biomaterials1997;18:56775.[28] Marchessault RH, Morehead FF, Walter NM. Liquid

crystal systems from fibrillar polysaccharides. Nature1959;184:6323.

[29] Lu Y, Wend L, Zhang L. Morphology and properties of soyprotein isolate thermoplastics reinforced with chitin whis-kers. Biomacromolecules 2004;5:104651.

[30] Paillet M, Dufresne A. Chitin whisker reinforced thermo-plastic nanocomposites. Macromolecules 2001;34:652730.

[31] Baxter A, Dillon M, Taylor KDA, Robert GAF. Improvedmethod for i.r. determination of the degree of N-acetylationof chitosan. Int J Biol Macromol 1992;17:1669.

[32] Hokugo A, Takamoto T, Tabata Y. Preparation of hybrid

scaffold from fibrin and biodegradable polymer fiber. Bio-materials 2006;27:6176.[33] Bunn P, Mottram JT. Manufacture and compression prop-

erties of syntactic foams. Composites 1993;24:56571.[34] Min B-M, Lee G, Kim SH, Nam YS, Lee TS, Park WH.

Electrospinning of silk fibroin nanofibers and its effect on theadhesion and spreading of normal human keratinocytes andfibroblasts in vitro. Biomaterials 2004;25:128997.

[35] Lv D, Cao C, Zhang Y, Ma X, Zhu H. Preparation ofinsoluble fibroin films without methanol treatment. J ApplPolym Sci 2005;96:216873.

[36] Kim U, Park J, Kim HJ, Wada M, Kaplan DL. Three-dimensional aqueous-derived biomaterial scaffolds from silkfibroin. Biomaterials 2005;26:277585.


MACROMOLEC

ULARNANOTECHNOL

OGY


13/13

[37] Freyman TM, Yannas IV, Gibson LJ. Cellular materials asporous scaffolds for tissue engineering. Prog Mater Sci2001;46:27382.

[38] OBrien FJ, Harley BA, Yannas IV, Gibson L. Influence offreezing rate on pore structure in freeze-dried collagen-GAGscaffolds. Biomaterials 2004;25:107786.

[39] Robert GAE. Chitin chemistry. Hong Kong: Macmillan;1992. p. 20367.

[40] Rovel J-F, Marchessaultf RH. In vitro chiral nematicordering of chitin crystallites. Int J Biol Macromol1993;15: 32935.

[41] Magoshi J, Mizuide M, Magoshi Y. Physical properties andstructure of silk. VI. Conformational changes in silk fibroininduced by immersion in water at 2 to 130C. J Polym Sci:Polym Phys 1979;17:51520.

[42] Asakura T, Kuzuhara A, Tabeta R, Saito H. Conforma-tional characterization of Bombyx mori silk fibroin in thesolid state by high-frequency carbon-13 cross polarization-magic angle spinning NMR, X-ray diffraction, and infraredspectroscopy. Macromolecules 1985;18:18415.

[43] Pearson FG, Marchessault RH, Liang CY. Infrared spectraof crystalline polysaccharide V. chitin. J Polym Sci 1960;43:10116.

[44] Chen M-H, Chen C-R, Hsu S-H, Sun S-P, Su W-F. Lowshrinkage light curable nanocomposite for dental restorativematerial. Dent Mater 2006;22:13845.

[45] Deng X, Hao J, Wang C. Preparation and mechanicalproperties of nanocomposites of poly(d,l-lactide) with Ca-deficient hydroxyapatite nanocrystals. Biomaterials 2001;22:286773.

[46] Kim H-W, Kim H-E, Salih V. Stimulation of osteoblastresponses to biomimetic nanocomposites of gelatinhydroxyapatite for tissue engineering scaffolds. Biomaterials2005;26:522130.

[47] Lee JH, Park TG, Park HS, Lee DS, Lee YK, Yoon SC,Nam J-D. Thermal and mechanical characteristics of poly-(l-lactic acid) nanocomposite scaffold. Biomaterials 2003;24: 277378.

[48] Lee YH, Lee JH, An I-G, Kim C, Lee D S, Lee YK, Nam J-D. Electrospun dual-porosity structure and biodegradationmorphology of Montmorillonite reinforced PLLA nano-composite scaffolds. Biomaterials 2005;26:316572.

[49] Favier V, Chanzy H, Cavaille JY. Polymer nanocompositesreinforced by cellulose whiskers. Macromolecules 1995;

28:63657.[50] Favier V, Canova GR, Shrivastava SC, Cavaille JY.

Mechanical percolation in cellulose whisker nanocompos-ites. Polym Eng Sci 1997;37:17329.

[51] Valk P, Pelt AWJ, Busscher HJ, Jong HP, Wildevuur CRH,Arends J. Interaction of fibroblasts and polymer surfaces:relationship between surface free energy and fibroblastspreading. J Biomed Mater Res 1983;17:80717.

[52] Ruardy TG, Schakenraad JM, Mei HC, Bussher HJ.Adhesion and spreading of human skin fibroblasts onphysicochemically characterized gradient surfaces. J BiomedMater Res 1995;29:141523.

[53] Qiu Q, Sayer M, Kawaja M, Shen J, Davies JE. Attachment,morphology, and protein expression of rat marrow stromal

cells cultured on charged substrate surfaces. J Biomed MaterRes 1998;42:11727.[54] Webb K, Hlady V, Tresco PA. Relationships among cell

attachment, spreading, cytoskeletal organization, and migra-tion rate for anchorage-dependant cells on model surfaces. JBiomed Mater Res 2000;49:3628.

[55] Schuler M, Owen GR, Hamilton DW, Wild M, Textor M,Brunette DM, Tosatti GP. Biomimetic modification oftitatium dental implant model surfaces using the RGDSP-peptide sequence: a cell morphology study. Biomaterials2006;27:400315.

[56] Pelham RJ, Wang YL. Cell locomotion and focal adhesionsare regulated by substrate flexibility. Proc Natl Acad SciUSA 1997;94:136615.

[57] Fereol S, Fodil R, Labat B, Galiacy S, Laurent VM, LouisB, Isabey D, Planus E. Sensitivity of alveolar macrophagesto substrate mechanical and adhesive properties. Cell MotilCytoskel 2006;63:32140.


MACROMOLEC

ULARNANOTECHNOL

OGY

crosslinked silk2

Documents