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Polymer Degradation and Stability 97 (2012) 955e963

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Polymer Degradation and Stability

journal homepage: www.elsevier .com/locate /polydegstab

Early stage structural evolution of PLLA porous scaffolds in thermallyinduced phase separation process and the corresponding biodegradabilityand biological property

Jundong Shao a,b,c, Cong Chen a,b, Yingjun Wang a,b,c, Xiaofeng Chen a,b,c, Chang Du a,b,c,*

a School of Materials Science and Engineering, South China University of Technology, Guangzhou 510641, ChinabNational Engineering Research Center for Tissue Restoration and Reconstruction, Guangzhou 510006, ChinacGuangdong Province Key Laboratory of Biomedical Engineering, South China University of Technology, Guangzhou 510006, China

a r t i c l e i n f o

Article history:Received 3 January 2012Received in revised form6 March 2012Accepted 9 March 2012Available online 17 March 2012

Keywords:Poly(L-lactic acid) scaffoldThermally induced phase separationStructural evolutionBiodegradabilityBiological property

* Corresponding author. School of Materials ScieChina University of Technology, Guangzhou 510641, Cfax: þ86 20 22236088.

E-mail address: duchang@scut.edu.cn (C. Du).

0141-3910/$ e see front matter � 2012 Elsevier Ltd.doi:10.1016/j.polymdegradstab.2012.03.014

a b s t r a c t

The structural evolution and the corresponding biodegradability and biological property of PLLA porousscaffolds during the early quenching period in a thermally induced phase separation process have beeninvestigated. The morphology, crystallization behavior, chemical structure, surface property, hydropho-bicity, biodegradability and biological property were studied by using SEM, WAXD, XPS, contact anglemeasurement, AFM, hydrolytic degradation and cell culture experiments. The initial phase separationresulted in an amorphous gel with the condensation of patches of amorphous precipitates, followed bythe nucleation of PLLA crystals. With extending the gelation process, a microporous structure wasformed. A temperature dependent phase behavior during the early quenching period has been observed.Along with the increase in the degree of crystallinity, structural transformation of the polymer towarda more ordered and compact state proceeded with the extending of the gelation time. The possiblesurface segregation of the methyl groups was confirmed by XPS analysis which may have certain effecton the increase of water contact angle. The evolution of architecture, crystallinity, chemical structure,surface property and the polymer chain packing mode, etc, during the early quenching period, hasa direct functional consequence in the hydrophobicity, biodegradability as well as biological property.

� 2012 Elsevier Ltd. All rights reserved.

1. Introduction

Nanofibrous porous scaffolds of various biocompatiblematerialshave been widely studied for applications in tissue engineering[1,2]. With a high porosity and a large surface area, these nano-fibrous porous scaffolds have long been noticed to be important forcell adhesion, proliferation, differentiation and the expression ofcell functions in tissue culture [3e6]. These make nanofibrousscaffolds ideal for applications in tissue engineering [7,8]. Tomimicking the size and scale of extracellular matrix (ECM)component, three major techniques are currently employed toproduce nanofibers from a wide variety of materials. Self-assemblyof peptide-amphiphile [9], block copolymers [10,11] and den-drimers [12] can typically create thinner fibrils with diameters ofa few to tens of nanometers. Electrospinning of polycaprolactone

nce and Engineering, Southhina. Tel.: þ86 20 22236062;

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(PCL), poly-lactic-co-glycolic acid (PLGA), poly(L-lactic acid) (PLLA)and other synthetic or natural polymers can routinely fabricatelarger nanofibers to micron scale fibers [13e15]. Phase separationprocess has been used to produce nanofibrous PLLA [16e19] andpolyhydroxyalkanoate (PHA) [20] porous scaffolds that mimic thearchitecture of natural ECM component such as the fibrillar struc-ture of collagen (50e500 nm in diameter) [21]. This latter approachis based on the thermodynamic demixing of a homogeneouspolymer-solvent system into a polymer-rich phase and a polymer-poor phase, usually by either cooling the solution below a binodalsolubility curve or exposure of the solution to an additionalimmiscible solvent. The thermally induced phase separation (TIPS)process is proposed to occur through spinodal liquideliquid phaseseparation and a consequential crystallization of the polymer-richphase [17,18], but the detailed mechanism is still not fullyunderstood.

PLLA has been attracting much attention from the academicviewpoint of structural interest as well as for practical applications.As one of the most prominent biodegradable and biocompatiblepolymer, PLLA is derived from 100% renewable resources and could

J. Shao et al. / Polymer Degradation and Stability 97 (2012) 955e963956

be eventually degraded into CO2 and H2O under natural conditions[22]. The morphology, crystallization process, crystal structure andconformational structure of the nanofibrous porous scaffolds areusually investigated by scanning electron microscopy, X-raydiffraction, differential scanning calorimetry and Fourier-transforminfrared spectroscopy [18,23]. As a semicrystalline polymer, thecrystallization behavior and crystal structure of PLLA have beenwidely studied. PLLA can form three kinds of crystal modifications(a-, b-, and g-form) [24e26] depending on the preparation condi-tions. Furthermore, a new crystal a0-form has been reported asa limited disorder crystalline modification of a-form. Pan et al.proposed that the a0-form crystal exists widely in the PLLA-basedproducts because of its low crystallization temperature [27,28].For all the tissue engineering applications, the biocompatibility ofsurface and the structural integrity of the scaffold are important fortissue regeneration to proceed. It is known that properties of PLLA,for instance, its biodegradability and biological property, greatlydepend on the morphological and structural characteristics.Understanding the structural evolution of PLLA scaffolds duringTIPS process and its relationship with the biodegradability andbiological property are of fundamental importance. In this study,we focused on the early stage of thermally induced phase separa-tion and gelation process by investigating the structural evolutionof PLLA scaffolds and their corresponding biodegradability andbiological property.

2. Materials and methods

2.1. Materials

Poly(L-lactic acid) with an inherent viscosity of 2.4 dl/g waspurchased from DaiGang Biomaterial Co. Ltd. (Shandong, China).Tetrahydrofuran (THF) was fromDAMAO REAGENT (Tianjin, China).Deionized water was obtained with a Milli-Q water filter systemfrom Millipore Corporation (France). All reagents were useddirectly without further purification.

2.2. Preparation procedures

The preparation of PLLA scaffolds through TIPS process followedthe procedures of Ma et al. [18]. In brief, a 4% (w/v) homogeneouspolymer solution was prepared by dissolving PLLA in THF andstirringwith amagnetic stirrer at about 60 �C for 2 h. The aliquots ofthe solutionwere rapidly transferred into a freezer at a temperatureof �24 �C (or �80 �C or liquid nitrogen) and kept for 1 min, 3 min,5 min, 10 min, 20 min and up to 2 h, respectively. Solvent exchangewas performed at 4 �C after quenching and the water was changedthree times a day for 2 days. The resultant sample was freeze-driedfor 48 h. The gelation point of 4% (w/v) PLLA/THF solution wasabout 25 �C [19] and the gelation occurred within several minutesat �24 �C [18].

A diluted 0.5% (w/v) PLLA/THF solutionwas prepared to cast 2-Dsamples on the glass slide for the AFM characterization. A series ofsamples with different quenching time (30 s, 1 min, 2 min, 5 min,10 min and 20 min) at �24 �C were obtained following the sameprocedures.

2.3. Scanning electron microscopy (SEM)

The morphology of 3-D scaffolds was observed by ScanningElectron Microscope (Quanta 200, FEI, The Netherlands) at15e20 kV by coating with gold for 120 s using a sputter coater (EM-SCD500, Leica, Germany).

Cells cultured on PLLA scaffolds were rinsed in PBS, fixed in 2.5%glutaraldehyde for 4 h at room temperature. Scaffolds were

dehydrated in a series of increasing concentrations of ethanol indistilled water (50%, 70%, 80%, 90%, 95%, 100%) for 60 min, 30 min,20 min, 10 min, 5 min and 5 min, respectively. The samples werethen sputter-coated with gold for 120 s and observed under a SEMat 10 kV.

2.4. Wide-angle X-ray diffraction (WAXD)

WAXD patterns were obtained by a D8 ADVANCE (Bruker,Germany), with Cu Ka radiation (l ¼ 0.1542 nm), working at 40 kVand 40 mA under the ambient conditions. A scan axis of 2q wasused to obtain diffraction patterns of a scan range between 5� and50�, with a scan rate of 0.2�/min.

2.5. X-ray photoelectron spectroscopy (XPS)

The XPS study was performed by using an Axis Ultra DLDspectrometer (Kratos, UK) with a monochromatic Al-Ka radiationsource (1486.6 eV). The analyzed spot sizewas 700� 300 mm. High-resolution scans of the C1s peak region (275e295 eV) were recor-ded with a pass energy of 40 eV.

2.6. Atomic force microscopy (AFM)

The surface topography was observed by an MFP-3D-S (AsylumResearch, USA) under AC mode (tapping mode) in an air atmo-sphere using Olympus AC160TS probe (Si3N4 cantilevers).

2.7. Roughness analysis

All the samples with different quenching timewere pressed intoa film for roughness analysis using Asylum MFP-3D softwaredeveloped on the Igor Pro 6.21 platform. The average roughness offilm surface was obtained from five randomly selected areas of50 mm � 50 mm with AFM height images of 256 � 256 pixelsresolution.

2.8. Contact angle measurement

The hydrophilicity of all samples was determined by employingstatic contact angle measurements with sessile drop method usingcontact angle goniometer OCA20LHT-TEC700-HTFC1500 (Data-physics, Germany).

2.9. Hydrolytic degradation

Hydrolytic degradation experiments of the PLLA scaffolds werecarried out in normal saline (NS, pH 6.95) and phosphate-bufferedsaline (PBS, pH 7.58) at 37 �C for a total period of 21 days. Aliquots ofPLLA samples (27e30 mg) were immersed in 50 ml solution for thepredetermined degradation time intervals.

2.10. Cell culture and seeding

Themouse embryonic carcinoma-derived chondrogenic cell lineATDC5 was purchased from Cell Bank, RIKEN BioResource Center(Tsukuba, Japan). The cells were cultured in L-DMEM supplementedwith 10% fetal bovine serum (FBS) in a humidified incubator at 37 �Cwith 5% CO2. The PLLA scaffolds were sterilized by soaking in 70%ethanol for 30 min, followed by soaking in PBS for 1 h, and washingwith cell culture medium for 1 h on an orbital shaker. Each scaffold(diameter of 8 mm and thickness of 2 mm) was seeded with 1�104

cells with a culture medium volume of 20 ml. After incubation for2 h, the cell-scaffold constructs were transferred into 24-well plate

J. Shao et al. / Polymer Degradation and Stability 97 (2012) 955e963 957

and 1 ml cell culture mediumwas added to each well. The mediumwas changed every 24 h afterwards.

2.11. Cell proliferation assay

Cell proliferation was examined using MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide) assay.The absorbance was determined at 490 nm under a microplatespectrophotometer (Varioskan Flash 4.00.53, Finland) and used asan indicator of cell proliferation.

2.12. Statistical analysis

All data were presented as means � standard deviation (SD). Inorder to test the significance of the observed differences betweenthe study groups, an unpaired Student’s t-test was applied. A valueof p < 0.05 was considered to be statistically significant.

3. Results and discussion

3.1. Morphology of the PLLA scaffolds

Fig. 1 shows scanning electron microscopy (SEM) images ofvarious PLLA scaffolds at predetermined quenching time intervals

Fig. 1. SEM images of the various samples with different quenching time at different(d) 1 min, �80 �C; (e) 3 min, �80 �C; (f) 5 min, �80 �C; (g) 1 min, LN; (h) 3 min, LN; (i) 5

(1 min, 3 min, 5 min) under different gelation temperatures(�24 �C, �80 �C and LN). After quenching for 1 min, the sample ata gelation temperature of �24 �C exhibited a relatively densemorphology with some platelet-like structure embedded in anamorphous matrix (Fig. 1a). When the quenching time increased to3 min and 5 min, a similar but more uniform platelet-likemorphology and an irregular fibrous structure were observed(Fig. 1b and c). At a gelation temperature of �80 �C, both amor-phous and fibrous structures were formed after quenching for1 min (Fig. 1d). Then the fibrous structure became the primarystructure and then the only structure after quenching for 3 min(Fig. 1e) and 5 min (Fig. 1f), respectively. For the matrices formed ata lower gelation temperatures such as in liquid nitrogen, a three-dimensional nanofibrous network was formed after quenchingfor 1 min (Fig. 1g). The fiber diameter varied from several tens tohundreds nanometers which was similar to the collagen. With theincrease of the gelation time to 3 min and 5 min, the nanofiberstended to be more uniform (Fig. 1hei). The nanofiber diameterswere 183 � 93 nm (n ¼ 60) for 3 min sample and 171 � 63 nm(n ¼ 60) for 5 min sample, respectively. There were no significantvariations in morphology when the gelation time was longer than5 min.

Fig. 2 shows the cooling profile of 4% (w/v) PLLA/THF solution ata gelation temperature of �24 �C, �80 �C and LN. The lower the

gelation temperatures: (a) 1 min, �24 �C; (b) 3 min, �24 �C; (c) 5 min, �24 �C;min, LN.

Fig. 2. The cooling profile of 4% (w/v) PLLA/THF solution at a gelation temperatureof �24 �C, �80 �C and LN.

J. Shao et al. / Polymer Degradation and Stability 97 (2012) 955e963958

gelation temperature, the faster was the cooling rate of PLLA/THFsolution. The temperature of PLLA/THF solution at the pre-determined quenching time intervals (1 min, 3 min, 5 min)depended on the gelation temperature, and played an importantrole in the thermally induced phase separation process. Theobservation suggested a temperature dependent phase behaviorduring the early quenching period. As the cooling rates of PLLA/THFsolution at �80 �C and LN were too fast, it was difficult to investi-gate the early stages of TIPS process under these two conditions. Aseries of typical PLLA samples quenching for 1 min, 3 min, 5 min,10 min, 20 min and 2 h at �24 �C were thus selected for detailedinvestigations in terms of early stage structural evolution of PLLAscaffolds, their biodegradability and biological property by usingWAXD, XPS, AFM, contact angle measurement, hydrolytic degra-dation and cell culture experiment respectively.

3.2. WAXD patterns

The crystalline structure of PLLA with different quenching timewas further investigated by WAXD analysis (Fig. 3). The degree ofcrystallinity was calculated as the percentage of the scatteredintensity of the crystalline phase over the scattered intensity of thecrystalline and amorphous phase [29]. An increase in the degree of

Fig. 3. WAXD patterns of the samples after 1 min, 3 min, 5 min, 10 min, 20 min and 2 hgelation at �24 �C. The inset graph is the enlarged (206) reflection.

crystallinity of PLLA with quenching time can be noted (Table 1).This crystallization process would result in more chain entangle-ment and decrease chain mobility. For comparison, all the diffrac-tion patterns were normalized using the strongest (110)/(200)reflection intensity. Based on the two strongest reflections from(110)/(200) and (203), the crystalline phases were characterized asthe a-form (2q ¼ 16.8� and 19.1�) at 1 min and the a0-form(2q¼ 16.4� and 18.9�) at 10 min and thereafter [27,28], respectively.With the increase of the gelation time up to 2 h, the two strongestreflections shifted remarkably toward the lower 2q side and thenstabilized. The samples being quenched for 3 min and 5 min couldbe the mixture of a- and a0-phase. As reported previously, tworelatively weak peaks at 2q ¼ 14.9� and 22.4� for (010) and (211)reflections were the characteristic diffraction of the a-phase,whereas a quite small peak (inset graph) at 2q ¼ 24.6� for (206)reflection was observed only in the a0-phase [27,28,30].

The calculation of lattice spacings (d) of the (110)/(200) and(203) diffractions further confirmed the variation of phase struc-ture of PLLA with different gelation time. The values of d110/200 andd203 increased gradually with the gelation time (Fig. 4). Thedifferent gelation times determined the different temperaturesunder which the nucleation of PLLA crystals occurred from theamorphous gel. After quenching for 1 min, the temperature of thesolution dropped to about 15 �C, and the temperature furtherdecreased to about 0 �C after 2.5 min (Fig. 2). The results suggestedthat a-form crystal nucleated preferentially at higher temperature,while a0-form prevailed at lower temperatures. It has been reportedthat the a0-form is a limited disorder a-form with slightly differentchain conformation and chain packing mode [30]. The WAXDresults were consistent with the morphological observation. Themorphological study of PLLA crystals from melt preparation alsoshowed the lamellar morphology on a micrometer scale for the a-form crystal and fibrillar crystallites for the a0-form crystal [30,31].

3.3. XPS results

XPS analysis have been performed to reveal the surface struc-tures of these PLLA samples. The XPS study was performed witha monochromatic Al-Ka radiation source (1486.6 eV). The analyzedspot size was 700 � 300 mm. High-resolution scans of the C1s peakregion (275e295 eV) were recorded with a pass energy of 40 eV.The C1s peak was normalized, and the first subpeak was set ata binding energy of about 284.6 eV. All data were collected andanalyzed using XPS Peak 41 software. As shown in Fig. 5, the XPSC1s core-level spectra were fitted with three peak components at284.6 eV, 286.6 eV and 288.7 eV, which were assigned to CeC/CeH,CeO and C]O. The relative content of CeC/CeH functional groupson the surface of PLLA samples increased gradually with the gela-tion time. The possible surface segregation of the methyl groupswas confirmed by XPS analysis which allowed the determination ofthe chemical composition of the sample surface of about 10 nmdepth. The relative composition of the C1s of aliphatic carbon

Table 1The crystallinity, roughness, contact angle and XPS results of the samples withdifferent gelation time.

Gelationtime

WAXDcc (%)

Roughness (nm) Contactangle (�)

XPS [relative contents (%)]

CeC, CeH CeO C]O

1 min 28.4 � 6.0 125.70 � 14.15 91.9 37.17 38.78 24.053 min 32.7 � 4.1 67.07 � 6.02 109.8 37.60 38.23 24.175 min 38.8 � 1.5 96.99 � 21.61 115.7 38.18 35.59 26.2310 min 40.8 � 0.4 67.18 � 14.53 117.1 38.43 34.44 27.1320 min 42.8 � 1.2 76.36 � 3.73 128.9 40.09 33.87 26.042 h 53.1 � 5.4 86.37 � 8.23 132.9 42.28 32.75 24.97

Fig. 4. Lattice spacings estimated from (203) and (110)/(200) reflections.

J. Shao et al. / Polymer Degradation and Stability 97 (2012) 955e963 959

bonds or carbonehydrogen bonds increased with the increasinggelation time. This result further confirmed the changes in surfacestructures such as chain conformation and the chain packing ofPLLA samples during TIPS process.

3.4. AFM and roughness analysis

We further investigated the evolution of nanofibrous textureduring the early stages of TIPS process by atomic force microscopy(MFP-3D AFM, Asylum Research, Santa Barbara, CA). A diluted 0.5%(w/v) PLLA/THF solution was prepared to cast 2-D samples on the

Fig. 5. C1s XPS spectra of the samples after (a) 1 min, (b) 3 m

glass slide for the study. A series of samples with differentquenching time (30 s, 1 min, 2 min, 5 min, 10 min and 20 min)at �24 �C were obtained following the same procedures.Comparing to the 3-D samples, these samples had a higherquenching rate and thus represent the lower temperature behaviorof PLLA gelation. Fig. 6 shows the typical AFM height images andthe corresponding three-dimensional images of 30 s, 2 min and20 min samples, respectively. In general, patches of amorphousprecipitates prevailed within 30 s quenching (Fig. 6a) under ACmode (tapping mode) in water, which may be the morphology ofpolymer-rich phase during the early stage of phase separationprocess. An intermediate nanofibrous structure with abundantcrystalline grains (Fig. 6b) was formed within 2 min quenching.This result demonstrated that the crystallization of PLLA wasa decisive step of nanofiber formation. After quenching for enoughtime, a mature nanofibrous structure was obtained (Fig. 6c).

All the samples with different quenching timewere pressed intothin films for roughness analysis using Asylum MFP-3D softwaredeveloped on the Igor Pro 6.21 platform. The average roughness offilm surface was obtained from five randomly selected50 mm � 50 mm AFM height images with 256 � 256 pixels reso-lution. The average roughness of film surfacewasmeasured by AFMsoftware and the value was reported in Table 1. All the samplesshowed small roughness of less than 130 nm, which can be ignoredin terms of the effects of the porous surface morphology.

3.5. Contact angle measurement

Contact angle of water droplet was measured by sessile dropmethod using the samples for roughness analysis and the valuewasreported in Table 1. The water contact angle of the samples

in, (c) 5 min, (d) 10 min, (e) 20 min and (f) 2 h gelation.

Fig. 6. Typical AFM height images and the corresponding three-dimensional images of (a) 30 S (AC mode in water), (b) 2 min and (c) 20 min samples.

J. Shao et al. / Polymer Degradation and Stability 97 (2012) 955e963960

increased along with the gelation time, which were also clearlyshown in Fig. 7. The porous surface morphology played an impor-tant role in the increase of the hydrophobicity. However, in ourstudy, all the samples showed quite small roughness which can beignored concerning the effects of surface morphology for contactangle measurement. The hydrophobicity displayed a strongdependency on the surface chemistry. The possible surface segre-gation of the hydrophobic methyl groups during the gelationprocess may have certain effect on the increase of water contactangle.

Fig. 7. Typical images of contact angle measurement for (a) 1 min, (b) 5 min, (c) 20 minsamples.

3.6. Hydrolytic degradation

The hydrolytic degradation behaviors of the PLLA scaffolds weretraced in NS and PBS at 37 �C. The pH values of the solutions during

J. Shao et al. / Polymer Degradation and Stability 97 (2012) 955e963 961

the degradation process were monitored. At predetermineddegradation time intervals, the scaffolds were removed from themedium, rinsed with distilled water, and dried under vacuum. Theresidue weight percentages of the samples immersed in PBS fordifferent times were determined after the lyophilization of thedegraded samples. Residueweight (%)¼Wr� 100/Wo, whereWr isthe dry weight of PLLA after degradation and Wo is the initial

Fig. 8. (a) pH of NS, (b) pH of PBS and (c) residue weight of PLLA in PBS as a function ofthe degradation time.

weight of PLLA. As shown in Fig. 8, all of the PLLA scaffoldsunderwent obvious degradation. The pH change and the residueweight change during the degradation process showed similardownward trend. PLLA can degrade into non toxic byproducts ofmainly lactic acid, which caused the pH value to decrease. Thedifference between the samples of different quenching times wasevident, especially after a longer degradation process. With

Fig. 9. ATDC5 cells attached on the PLLA scaffolds with different quenching time(a) 1 min, (b) 5 min, (c) 20 min.

J. Shao et al. / Polymer Degradation and Stability 97 (2012) 955e963962

increasing quenching time, the degradation rate of PLLA scaffoldsgradually slowed down.

The hydrolysis of ester linkage was confirmed as the primarymechanism of degradation, followed by the subsequent reductionof molecular weight, to result in water-soluble oligomeric and/ormonomeric products [32]. The hydrolytic degradation performanceof polymers was affected by many factors, such as the architecture,crystallinity, chemical structure, degradation environment, surfaceproperty, and so on [33]. According to the WAXD results, thecrystallinity increased alongwith the quenching time, whichwouldresult in more chain entanglement and decrease chain mobility.This crystallization process negatively influenced the degradation.The scaffolds with lower crystallinity had a random chain packingstructure rendering it more amorphous, which would favor thehydrolytic degradation. The surface hydrophilicity was also animportant factor affecting the degradation rate. The contact angleof water showed a reverse tendency with the degradation rate. Thesample with a higher contact angle negatively influenced thehydrolytic susceptibility by steric hindrance and made it difficultfor the water to penetrate into the bulk so that hydrolysis wasretarded to some extent [34].

3.7. Morphology of cells on scaffolds and proliferation of cells

Fig. 9 shows the typical images of themorphology of ATDC5 cellson the PLLA scaffolds. After the incubation in L-DMEM supple-mented with 10% FBS for 24 h, the cells on nanofibrous scaffoldswith longer quenching time showed a better initial adhesion andspreading well with many small processes interacting with PLLAnanofibers (Fig. 9c). In contrast, the cells on the scaffolds onlyquenching for several minutes showed a spherical morphologywith few processes (Fig. 9aeb). We can conclude that the nano-fibrous scaffolds with longer quenching time were beneficial forcell adhesion.

Cell proliferation was examined using MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay onday 1, 4 and 7. Scaffolds with cells weremoved to a new 24-well cellculture plate. 1 ml of 0.5 mg/ml MTT in cell culture medium wasadded to each well and the constructs were incubated at 37 �C for4 h to allow the formation of purple formazan crystals. DMSO wasadded to dissolve the purple formazan crystals into a coloredsolution. The absorbancewas determined at 490 nm at amicroplatespectrophotometer (Varioskan Flash 4.00.53, Finland) and used asan indicator of cell proliferation. As shown in Fig. 10, a significant

Fig. 10. Proliferation of cells on different PLLA scaffolds for 1 day, 4 days and 7 days(*p < 0.05, **p < 0.01, ***p < 0.001 to the 1 min sample).

increase in cell number was observed on all scaffolds with pro-longed culture. After cultured for 4 days and 7 days, cell number onthe scaffolds showed an increasing trend along with the quenchingtime of PLLA. This suggested that the PLLA scaffolds with longerquenching times provided a suitable surface for cell attachmentand proliferation.

4. Conclusions

In summary, the early stage of PLLA porous scaffolds formationvia thermally induced phase separation process involved thecondensation of patches of amorphous precipitates and a gradualcrystallization from the gel. With extending the gelation process,a microporous structure can be formed. A temperature dependentphase behavior during the early quenching period occurred. The a-form crystal appeared after quenching for 1 min, corresponding toa gel temperature around 15 �C. After quenching for sufficient timeto decrease the gel temperature below 0 �C, a limited disorder a0-form crystal prevailed and the content of a0-phase in the mixturewas increasing gradually with the gelation time. Along with theincrease in the degree of crystallinity, structural transformation ofthe polymer toward a more ordered and compact state proceededwith the extending of the gelation time. The evolution of archi-tecture, crystallinity, chemical structure, surface property and thepolymer chain packing mode, etc., during the early quenchingperiod, had a direct functional consequence in the hydrophobicity,biodegradability as well as biological property.

Acknowledgments

The work is supported in part by National Basic ResearchProgram of China (2012CB619100, 2011CB606204), Program forNew Century Excellent Talents in University (NCET-08-0210),Program for Changjiang Scholars and Innovative Research Team inUniversity (IRT 0919) and the Fundamental Research Funds for theCentral Universities (2009ZZ0004, 2011ZG0001).

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