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Acta Biomaterialia 4 (2008) 950–959

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Dynamic mechanical behavior of starch-based scaffolds in dryand physiologically simulated conditions: Effect of porosity

and pore size

Satyabrata Ghosh a,b, Victor Gutierrez a,b, Carolina Fernandez a,b,Miguel A. Rodriguez-Perez c, Julio C. Viana d, Rui L. Reis a,b, Joao F. Mano a,b,*

a 3Bs Research Group (Biomaterials, Biodegradables and Biomimetics), Department of Polymer Engineering, University of Minho, Braga 4710-057, Portugalb Institute for Biotechnology and Bioengineering (IBB), PT Government Associated Laboratory, Braga, Portugal

c Department of Condensed Matter Physics, University of Valladolid, E-47011, Valladolid, Spaind Institute for Polymers and Composites (IPC), Department of Polymer Engineering, University of Minho, Guimaraes 4800-058, Portugal

Received 27 October 2007; received in revised form 28 January 2008; accepted 4 February 2008Available online 15 February 2008

Abstract

The three-dimensional scaffolds of a blend of starch and poly(L-lactic) acid, SPLA70, were produced using compression molding ofpolymer/salt mixture followed by leaching of salt. One series of scaffolds were prepared with varying polymer-to-salt ratio while keepingthe salt size constant, and the other series of scaffolds were prepared with varying salt sizes while keeping the polymer-to-salt ratio con-stant. The X-ray microcomputed tomography and scanning electron microscopy assay were used to analyze the porous morphologies,porosity and distribution of porosity of the porous scaffolds. Salt-free and integrated SPLA70 scaffolds with porosities ranging from 74%to 82% and pore sizes of 125–250 to 500–1000 lm can be fabricated using the present fabrication technique. The water uptake of theSPLA70 scaffolds increases with increasing porosities and also with increasing pore size. In dry state, the storage modulus decreases withincreasing porosity and also with increasing pore size. The normalized modulus values are related to normalized density of the scaffoldsby a power-law function with an exponent between 2 and 3. For the immersed scaffolds under physiological conditions, the storage mod-ulus was less dependent on porosity and pore size. However, the loss factor increased significantly compared with dry state measure-ments. The present study clearly shows that the mechanical performance of porous polymeric constructs in dry and in immersedstate is completely different, and for comparison with biomechanical performance of tissues, the tests should ideally be performed inimmersed state.� 2008 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Keywords: Dynamic mechanical properties; Porosity; Pore size; Dry and immersed state; Starch blend

1. Introduction

Tissue engineering approaches have great potential inthe biological and functional regeneration of tissues, allow-ing for overcoming the lack of donor tissue and to promotea biologically and mechanically functional tissue. An

1742-7061/$ - see front matter � 2008 Acta Materialia Inc. Published by Else

doi:10.1016/j.actbio.2008.02.001

* Corresponding author. Address: 3Bs Research Group (Biomaterials,Biodegradables and Biomimetics), Department of Polymer Engineering,University of Minho, Braga 4710-057, Portugal. Tel.: +351 253 604 497.

E-mail address: jmano@dep.uminho.pt (J.F. Mano).

important step to engineer tissues is the development ofporous three-dimensional scaffolds for different anatomicallocations in the body. Generally, the materials of scaffoldsare either of natural origin or synthetic biodegradable poly-mers [1–4]. Synthetic polymers have design flexibilities interms of materials composition, processability, controlover macro- and microstructures, and mechanical proper-ties [4]. These properties can be tuned for specific applica-tions. However, natural polymers are also used inbiomedical applications for their excellent biocompatibilityand biodegradability [3,5].

vier Ltd. All rights reserved.

S. Ghosh et al. / Acta Biomaterialia 4 (2008) 950–959 951

Poly(a-hydroxy acids) including poly(lactic acid) (PLA),poly(glycolic acid) (PGA) and their co-polymers (PLGA)are the widely accepted polymers for most of the tissueengineering applications [6]. These thermoplastic polyestershave reasonable biocompatibility, biodegradability,mechanical strength and easy processability. Blends ofPLLA and starch (SPLA) have also been proposed in ourgroup to be used in the biomedical field, and were foundfrom cell adhesion and proliferation tests, to interact posi-tively with cells [7].

The fabrication of porous scaffolds from a polymer meltallows fast production of scaffolds with different shape andsizes in an efficient way. The most straightforward way is tomelt a polymer of a polymer/salt mixture, cool it and sub-sequently remove the salt particles, thus producing the por-ous scaffolds. The desired porosity and pore sizes could beindependently controlled by the fractions and sizes of saltparticles, respectively [8,9].

The mismatch between the mechanical properties ofscaffolds and the tissue intended to be regenerated maycompromise the clinical success of the tissue engineeringconstruct. In this regard, stiffness is a relevant mechanicalproperty of scaffolds as it will determine the deformabilityof the structure upon implantation. The modulus of elastic-ity describes the slope of the stress–strain relationship for agiven material under load. Such property depends stronglyon the porosity in the case of a porous system, and mayalso be influenced by pore size [10]. In the case of poly-meric-based materials, one should also consider their visco-elastic properties as they will determine the capability ofthe device to dissipate mechanical energy and are responsi-ble for the time-dependent nature of its mechanical perfor-mance. Moreover, living tissue also exhibits a clearviscoelastic behavior and, ideally, the implantable scaffoldshould present similar properties in order to maintain thecontact with the surrounding tissue during all the physio-logical stress–strain history. Of course, this would be diffi-cult to achieve using polymer-based materials intended tobe used in the replacement or regeneration of hard tissues.Although some rheological properties could be similar inbiomaterial and tissue (e.g. damping), stiffness-relatedproperties are more difficult to achieve.

Dynamic mechanical analysis, DMA, is a non-destruc-tive technique widely used in the characterization of theviscoelastic properties polymer-based systems includingbiomaterials covering wide temperature and frequencyranges [11]. Such studies are particularly relevant if thetests are performed at physiologically meaningful condi-tions, i.e., with the specimens completely incubated in solu-tions at 37 �C. Similar procedure was previouslyimplemented to study starch-based biomaterials [12,13].However, such dynamic tests performed at conditions closeto the in vivo situation are not yet sufficiently establishedamong the community. This work intends to strengthenthe importance of such kinds of experiments.

In this study, the biodegradable polymeric scaffolds wereprepared using compression molding technique with the

melting of the polymer in a polymer/salt mixture followedby the dissolution of salt in water. One series of scaffoldswas prepared by varying the polymer-to-salt ratio whilekeeping the salt size constant, and the other series of scaf-folds was prepared by varying the sizes of the salt particleswhile keeping the polymer-to-salt ratio constant. The mor-phology of the scaffolds was assessed by X-ray microcom-puted tomography analysis and scanning electronmicroscopy. The accessibility of internal pores or hydraulicconductivity of the scaffolds prepared with different poresizes and porosities was determined by relative wateruptake of the scaffolds. Moreover, the dynamic mechanicalproperties of the scaffolds were assessed in both dry andincubated state at physiological temperature of 37 �C.The relationship between mechanical properties, pore sizesand porosities were then established.

2. Materials and methods

2.1. Materials and scaffold processing

A 30/70 (wt.%) blend of starch and PLLA (SPLA70)was used in this study. The SPLA70 was obtained by meltblending of 70 wt.% PLLA and 30 wt.% of corn starch. ThePLLA of SPLA70 had L-lactide content of 94%.

2.1.1. Powder preparation and compounding

SPLA70 pellets were dried at 50 �C in a vacuum ovenand cryogenically milled in an ultra-centrifugal mill (Rets-ch, ZM-100) with liquid nitrogen at 14,000 rpm. Theselected NaCl particle sizes were hand-sieved with thestacked stainless steel sieves (ISO3310, Endecotts Ltd.,England).

2.1.2. Compression molding

The compression-molded disks of around 5 mm thick-ness and 80 mm diameter were prepared on a steel moldusing a Moore hydraulic press (UK), with 50 ton capacity.Prior to compression molding, the powdered SPLA70 wasdried in a vacuum oven. The polymer/salt mixtures wereput in between Teflon release papers in the mold. The moldwas then placed in between the hot plates of the hydraulicpress at top and bottom platen temperature of 180 �C. Toremove the trapped air in bulk powder, the pressure wasslowly raised around 20 MPa and released, and this processwas repeated five times. The polymer/salt mixture was thenallowed to melt for 6 min at constant pressure of 151 kPa,the weight of top half of the mold. The mold was thencooled to room temperature. The initial compositions ofthe constituents that were used to prepare the studied scaf-folds are shown in Table 1.

2.1.3. Porogen leaching

The molded disks were sliced to �5 � 5 � 5 mm3

cuboids and put into water. The leaching was performedin beakers filled with de-ionized water at 37 �C. Waterwas replaced every 4 h. The presence of NaCl content

Table 1Porous SPLA70 scaffolds prepared by compression molding of SPLA70and NaCl mixtures followed by salt leaching

Scaffolds NaCl size (lm) NaCl fraction Porosity

(wt.%) (vol.%) (%)

A1 250–500 70 59 73.9A2 250–500 80 70 78.6A3 250–500 85 78 82.0B1 125–250 80 70 80.2B2 250–500 80 70 78.6B3 500–1000 80 70 77.9

A2 = B2.

952 S. Ghosh et al. / Acta Biomaterialia 4 (2008) 950–959

was checked by silver nitrate solution acidified with dilutenitric acid. The leaching protocol was continued till 48 h.

The pyrolysis of the porous scaffolds was performed in amuffle furnace. The scaffolds were dried (in a vacuum ovenfor 24 h at 50 �C followed by cooling in a desiccator),weighed and placed in a crucible that was previouslycleaned and dried. The crucible containing the scaffoldwas then placed inside the furnace. The temperature wasraised slowly to 400 �C and kept for 5 min, followed to700 �C for 35 min. Thereafter, the furnace was turned off,allowed to cool till 50 �C. The crucible was taken out, keptin a desiccator for 24 h at room temperature and weighed.The wt.% residue was calculated as

Residual salt content ¼ mp

md

� �� 100 ð1Þ

where mp and md are the weights of residue after pyrolysisand dried scaffold, respectively.

2.2. Characterization of the scaffolds

2.2.1. Porosity and water uptake

The porosities of the scaffolds were determined by X-raymicrocomputed tomography (microCT) using a Sky-Scan1072 equipment (Belgium). The porous scaffolds weremounted on a rotary stage and scanned under a 40 kVsource for a complete rotation of 360�, during which 300slices were produced. The resolution obtained was8.79 lm pixel�1.

The scaffolds were immersed in water and a series ofshort evacuation–repressurization cycles were carried outto force water into the pores of the scaffolds. The scaffoldswere kept in water for 48 h to ensure the water impregna-tion into the open pores. The water uptake of the porousscaffolds was calculated as

water update ¼ mw � md

md

� �� 100 ð2Þ

where mw is the weight of wet scaffold.

2.2.2. Morphological characterization

Porous morphologies of the scaffolds were examinedusing scanning electron microscopy (SEM) (Leica Cam-bridge S-360, UK) at 15 kV. The specimens were coated

with gold using a sputter coater (JFC 1100, Jeol). Theinternal porous morphologies were characterized usingmicroCT analysis.

2.2.3. Dynamic mechanical analysis

In dynamic mechanical analysis the specimens aresubjected to a sinusoidal load, with a rate defined by afrequency f (in cycles s�1 or Hz) or an angular frequencyx = 2pf (in rad s�1): r ¼ r0 expðixt þ dÞ, where i = (–1)1/2.The response (a strain) though sinusoidal is not in phasewith the developed stress, and lags behind the stress bysome phase angle d between 0 and 90�. In this case, themodulus has a complex nature:

E�ðxÞ ¼ d�¼ ðr0=�0Þ expðidÞ ¼ ðd0=�0Þðcos dþ i sin dÞ

¼ E0 þ iE00 ð3Þ

The storage modulus, E0, is the elastic or real componentof complex modulus, E*, and is in phase with r. The stor-age modulus is related to the amount of energy stored bythe material during a cycle. The loss modulus, E00, is the vis-cous component of E*, and is p/2 out of phase with thestress, and is associated with the dissipation of energydue to internal friction at the microscopic level. The lossfactor, tan d ¼ E00=E0, is a measure of the damping capabil-ity of a material.

The dynamic mechanical analysis (DMA) was per-formed on the porous three-dimensional scaffolds undercompression in a TRITEC 2000B DMA (Triton Technol-ogy, UK). The dry state measurements of the porous scaf-folds with dimensions around 5 � 5 � 5 mm3 were carriedout at a physiological temperature of 37 �C. The tests wereperformed under compression from 0.05 to 50 Hz at a con-stant dynamic displacement of 50 lm. Five points weremeasured within each decade.

For the measurements at the wet state, a phosphate buf-fer solution (PBS) was forced into the scaffolds by briefevacuation–repressurization cycles and kept in a soakedcondition for 45 min prior to placing it into PBS bath forthe DMA experiments. The measurements were carriedout under compression by immersing the wet scaffolds inPBS bath at a constant physiological temperature of37 �C. The temperature was equilibrated by keeping thescaffolds 15 min in the PBS bath of DMA. The tests wereperformed under compression in the frequency range of0.05–10 Hz at a constant dynamic displacement of 50 lm.For each individual scaffold, the data is averaged over threeconsecutive runs. Three samples were measured for eachtype of scaffolds.

3. Results and discussion

3.1. Scaffold characterization

The three-dimensional porous scaffolds can be conve-niently prepared by compression molding of thermoplastic

S. Ghosh et al. / Acta Biomaterialia 4 (2008) 950–959 953

polymer powder and salt particulate mixtures followed bythe removal of salt fraction. In the compression moldingtechnique, the sintering of bulk polymer/salt mixture intoa homogeneous compact form through melting dependson the viscosity of the molten polymer. The melting tem-perature of this PLLA in SPLA70 was �155 �C [14]. How-ever, the porous three-dimensional scaffolds prepared inthe temperature range of 155–170 �C were not homoge-neous due to limited fluidity of the molten polymer. Theviscosity of PLLA is sensitive to temperature and decreasesrapidly with increasing temperature [15]. Moreover, bothPLA and starch are sensitive to degradation at elevatedtemperature. The molding temperature of 180 �C and resi-dence time of 6 min are a compromise between degradationand fluidity of PLLA that allowed producing homogeneousscaffold with minimal degradation of the constituentpolymers.

With the addition of different fraction of salt particu-lates, the scaffold porosity can be tuned. In this case, theconcern arises with the minimum and maximum fractionsof salt that can be added to obtain stable scaffolds withminimum or no salt content. Typically, the maximumpacking of mono-dispersed dense spheres is 68 vol.%. Inthe present study, starting from 60 wt.% with a step of10 wt.%, the maximum salt concentration of 90% wastested. Below 70 wt.% of salt fraction, the salt particles can-not be fully extracted. The porous scaffolds prepared with

Fig. 1. Representative two-dimensional microCT images showing the homogenmicroCT reconstruction of three-dimensional scaffolds. The scale bar = 2 mm

salt fraction of 90 wt.% are prone to disintegration. Thestable scaffolds were achieved with 85 wt.% of pre-seededsalt particulate. The fabrication procedure for all the scaf-folds were maintained the same.

One series of scaffolds was prepared with increasingporogen fraction while maintaining the constant pre-seededsalt size (Table 1). Fig. 1 shows that the distribution ofpores was uniform throughout the matrix. The microCTimages in Fig. 1 show that the number of pores is increas-ing from left (A1) to right (A3), as expected. The secondseries of scaffolds are prepared with varying pore sizeswhile the porogen fraction was kept constant. As expected,Fig. 1 shows that the pore sizes are increasing from top(B1) to bottom (B3) with increasing porogen sizes.

The SEM micrograph in Fig. 2 shows the pore size andpore wall distribution on a higher magnification. The num-ber of pores is increasing from left to right, i.e. withincreasing porogen fraction. However, with the higher frac-tion of salts, there are some pores larger than the size ofsalts. This is expected as salt–salt contact points increaseswith more than 68 vol.% pre-seeded particles; in such casessome of the resulting agglomerates will give rise, uponleaching, to pore sizes bigger than the pre-seeded salt size.

It is clearly visible in the higher-magnification SEMmicrographs in Fig. 2 that the pore sizes are increasingfrom top to bottom, i.e. with increasing pre-seeded salt sizeat constant fraction of salt. It has been frequently observed

eous pore distribution across the porous scaffolds, the insets represents the.

Fig. 2. Representative SEM micrographs of the scaffolds showing the increase of porosities along the horizontal direction prepared with constant salt sizeof 250–500 lm and increase of pore sizes along the vertical direction prepared with constant salt fraction of 80 wt.%.

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with big salt sizes, that a number of small pores (5–10 lm)appear on the pore wall and the pore wall thickness wasnon-uniform. The smaller pores in the pore walls may offeradvantages in tissue engineering application, as it increasesthe interconnectivity.

Fig. 3 shows that the apparent porosities were increasedfor all the scaffolds with respect to the pre-seeded salt frac-tion. The difference between apparent porosities and pre-seeded salt fractions were decreased with increasing saltfraction. The possible reason for the higher apparentporosity compared with pre-seeded salt fraction could bethe loss of poorly integrated polymeric fragments duringleaching of porogen. Similar results were obtained withPLLA, SPLA50 (blend of 50:50 wt.% starch and PLLA),composites of PLLA/hydroxyapatite porous compositesprepared with compression molding and salt leaching tech-nique [16,17].

An increase in apparent porosities was also observedwith scaffolds prepared with increasing salt size at constantfraction of pre-seeded salt particulates. Surprisingly, theresults show that the apparent porosities are decreasingwith increasing pre-seeded salt size. Similar results werealso reported with poly(ether ester) copolymer scaffoldsprepared with compression molding and salt leaching tech-nique [18]. Nevertheless, the variation of porosity when dif-ferent salt particle sizes are used is small (77.9–80.2% in theextremes), when compared with the changes in pore sizes.Therefore we can consider that any change observed in thisset of scaffolds, B1, B2 and B3, will be mainly a conse-quence of the pore sizes.

Fig. 4 shows the porosity distribution across the scaf-folds obtained from the analysis of the microCT results.The porosity distribution is quite uniform throughout thescaffold. The distribution is narrower with both the

increased pre-seeded salt sizes and salt fractions. Theporosity distribution is broader with lowest fraction(70 wt.%) of pre-seeded salt fraction (A1).

The porous scaffolds prepared with 70–85 wt.% of pre-seeded NaCl particulates do not contain residual salt parti-cles after NaCl leaching in water. This has been verifiedfrom the pyrolysis of the porous scaffolds. This result indi-cates that compression molding and salt leaching techniqueallows preparing salt-free and stable three-dimensionalstarch-based polymeric scaffolds with 74–82% porosity.

Fig. 5 shows that the SPLA70 scaffolds with the samepore size take up 217–366% of water (a variation of 69%)with increasing porosities. The average hydraulic conduc-tivities of porous SPLA70 scaffolds are higher than inPLLA scaffolds of similar porosities [16]. This could bedue to the presence of hydrophilic starch that assists touptake more water than the hydrophobic PLLA fraction,contributing to an increase of the total water uptake forSPLA70 scaffolds. As expected, the water uptake followsthe same trend of porosities for A1, A2 and A3 scaffolds.The corresponding porosities of A1 and A3 were 73.9and 82%, respectively (a variation of 11%). So, the incre-ment in water uptake is substantially higher than the incre-ment in porosity. The hydraulic conductivity increasessignificantly with the increasing of porosities from 73.9%to 82%. The higher water uptake compared with the incre-ment of porosity has been reported with other polymericscaffolds prepared with compression molding and saltleaching technique [19].

For B1, B2 and B3 scaffolds, the water uptake increaseswith increasing pore size. The average water uptake of B3scaffolds (pore size 500–1000 lm) was 380%, and B1 scaf-folds (pore size 125–250 lm) had 239% (a variation of59%). Note that the B3 scaffolds had slightly lower porosity

Fig. 3. Comparison between the apparent porosities obtained with respectto the fraction and size of the pre-seeded salt particles. The dotted linerepresents the theoretical porosity.

Fig. 4. Representative porosity distribution along the thickness of theporous scaffolds: (a) effect of pre-seeded salt fractions at constant salt sizeof 250–500 lm, and (b) varying salt sizes at constant salt fraction of80 wt.%.

Fig. 5. Variations of equilibrium water uptake with the variation ofporosity and pore size of SPLA70 scaffolds at 37 �C and at atmosphericpressure.

S. Ghosh et al. / Acta Biomaterialia 4 (2008) 950–959 955

than B1 scaffolds (a variation of �3%). The average saltparticle sizes of B1 scaffolds were nearly four times lessthan the corresponding salt particle sizes of B3. Therefore,the theoretical pore surface area of B1 scaffolds is aroundfour times higher than B3 scaffolds. If the pore surface areais accountable for the water uptake of the scaffolds, itshould follow the order B1 > B2 > B3. However, the wateruptake of the scaffolds follows B3 > B2 > B1 (see Fig. 5).The reason for the discrepancy is likely due to trappedair in the bulk pores, which could be increased withincreasing surface areas of decreasing pore sizes. Thesevoids may not allow water to permeate into the pores, thusresults a lower uptake with decreasing pore size. Moreover,it could be clearly evident from the micrographs of B2 andB3 in Fig. 2 that the number of small pores in the walls sep-arating the bigger pores created by dissolution of salt par-ticles are increasing with increasing salt sizes. Thisperforation in pore walls may have facilitated the hydraulicconductivity that led to the higher water uptake of the scaf-folds with bigger pores.

3.2. Dynamic mechanical properties

The solid-state rheological properties of the starch-based scaffolds with different porosities and pore sizes weredetermined by DMA. The storage modulus and the lossfactor of these two sets of scaffolds are shown as a functionof frequency in Fig. 6, where the measurements were per-formed in dry state. No evident variations along the fre-quency axis are seen in these curves, indicating that theviscoelastic properties of the scaffolds are quite steadywithin a large time scale range; this suggests that no dis-crete relaxation process takes place in this frequency win-dow at 37 �C. The possible reason for the steadyviscoelastic performance of the scaffolds could be due tothe fact that the material was studied at 37 �C, and its glasstransition temperature is about 60 �C [14].

The values found in Fig. 6 for a frequency of 1 Hz areshown in Fig. 7, as a function of porosity. The results vary

Fig. 6. Variation of E0 (filled symbols) and tand (open symbols) withfrequency for the different porous SPLA70 scaffolds. The experimentswere carried out at 37 �C and at dry state. (a) Effect of porosity on thescaffolds prepared with constant salt size of 250–500 lm (A1 = N,A2 = d, A3 = j); (b) effect of pore size on the scaffolds with constantsalt fraction of 80 wt.% (B1 = N, B2 = d, B3 = j).

Fig. 7. (a) Variation of E0 with porosity (d) and pore size (j) of theSPLA70 scaffolds at 1 Hz. (b) Variation of tand with porosity (d) andpore size (j) of the SPLA70 scaffolds at 1 Hz. All the experiments werecarried out at 37 �C and at dry state.

956 S. Ghosh et al. / Acta Biomaterialia 4 (2008) 950–959

from 5 MPa to 45 MPa, depending on the structure. Forcomparison, the flexural storage modulus at 37 �C mea-sured at 1 Hz for compact SPLA70 was found to be1400 MPa [12], i.e. around two orders of magnitude higherthan the scaffolds. As expected, the storage modulusdecreases with increasing porosity for the group A1, A2,A3 (Fig. 7a). However, the results from the group B1,B2, B3 also allow us to conclude that the pore size playsan important role in the stiffness of the scaffolds: for nearlythe same porosities, the storage modulus decreases withincreasing pore size. The similar trend was observed withscaffolds prepared with salt leaching techniques but withother materials [18]. The values of the loss factor atf = 1 Hz in Fig. 7b indicates that in general most of the val-ues do not correlate with either porosity or with pore size.Using a basic viscoelastic model considering a porous sys-tem as a combination in parallel of a solid material, corre-sponding to the matrix, coupled with an empty space,corresponding to the pores, this should be expected, wherethe loss factor of the ensemble is the one of the matrix. In

fact, the loss factor of the compact material was found tobe 0.028, at 37 �C and 1 Hz [12], which is similar to mostof the values in Fig. 7b. However, it is clear that, for A3,the tand value is significantly higher compared with otherscaffolds; this also happens in the entire frequency rangeexplored (see Fig. 6). This behavior should be explainedby other dissipation mechanisms that occur preferentiallyin such specimens with higher porosity.

One possibility could be a friction effect of the perfora-tion of the pore walls, frequently observed in higher poros-ity scaffolds of A3 (Fig. 2) compared with compact surfacesof A1 (Fig. 2). It would be also interesting to compare theobtained values of tand with those found in tissues. Forexample, Buechner et al. [20] collected a series of resultsof tand of bone in a frequency range of 10�7–107 Hz.The values of the loss factor ranged between 0.01 and0.1, but for frequencies physiologically felt by living bone,say 10�2–103 Hz, the values for human bone varies typi-cally between 0.02 and 0.04. This interval is in agreementwith the order of magnitude of the values of tand foundin the studied scaffolds, not only at 1 Hz but in all fre-quency ranges explored (see Fig. 6).

Fig. 8. Variation of E0 (filled symbols) and tand (open symbols) withfrequency for the different porous SPLA70 scaffolds at 37 �C, measured inPBS. The experiments were carried out at 37 �C and in immersed state. (a)Effect of porosity on the scaffolds prepared with constant salt size of 250–500 lm. (A1 = N, A2 = d, A3 = j); (b) Effect of pore size on the scaffoldswith constant salt fraction of 80 wt.% (B1 = N, B2 = d, B3 = j).

S. Ghosh et al. / Acta Biomaterialia 4 (2008) 950–959 957

As the modulus of a porous scaffold is related to themodulus of parent material and porosity, it is reasonableto compare the normalized modulus, i.e. the ratio of themodulus of the porous scaffold with the modulus of com-pact material, with normalized density, i.e. the ratio ofthe apparent density of the porous scaffold with the densityof compact material. For porous construct, the relationshipbetween normalized modulus and normalized density isrelated by the following expression [21]:

EEc¼ C

qqc

� �n

ð4Þ

where E and Ec are the moduli of porous scaffold and solidmaterial, respectively; q and qc are the densities of porousscaffold and solid material, respectively. The constants C

and n depend on the microstructure of the scaffold. Theconstant C includes all geometric constants of proportion-ality. The value of n generally lies in the range of 1 < n < 3,resulting a wide range of values for modulus at a given den-sity [18]. For an isotropic open cell structure, the exponentn is expected to be 2. A number of factors affect modulus–porosity relationship, such as open and closed pores; thedistribution and dispersion of pores; and the shape and ori-entation of pores. The dependence of C and n on the micro-structure of highly porous scaffolds with randomdistribution of pores, such as the ones investigated in thepresent study, is not well understood.

The values of n were calculated from Eq. (4) by putting theexperimental values of moduli for all the SPLA70 scaffolds at1 Hz, the modulus of compact SPLA70 as 1400 MPa [12] andC = 1. The n values for A1, A2 and A3 scaffolds were 2.53,2.45 and 2.96, respectively; and for B1, B2 and B3 scaffoldswere 2.22, 2.45 and 2.69, respectively. Thus the values of n

increase with increasing porosities and also with increasingpore sizes. The experimental data fits reasonably well withthe theoretical model with the exponent values 2 < n < 3 withC = 1, and is appropriate to describe the elastic modulus ofporous scaffolds [18,22]. Such an approach could enablescaffolds to be designed with a given stiffness adequate fora desired tissue engineering application.

DMA experiments were also performed on samples inimmersed state, in order to estimate the viscoelastic prop-erties of the specimens in more realistic physiological con-ditions. The results obtained at 37 �C are shown in Fig. 8,where the two sets of samples are shown separately. Theviscoelastic properties are quite steady along the fre-quency range analyzed, although the tand values inimmersed state tend to decrease more with increasing fre-quency compared with the dry state measurement. Espe-cially for the samples with higher porosity, it is foundthat the storage modulus is similar to the dry samples.This is somewhat unexpected as the material can takeup some quantity of water and the stiffness is expectedto decrease upon immersion. However, in the case ofimmersed samples the pores are filled with water thatcould render more difficult the deformability of the scaf-fold through a hydrostatic effect.

The values of tand for the wet samples are higher thanfor the dry samples (Fig. 6). The contribution of the fric-tion between the water molecules and the construct couldbe responsible for this increment in the loss factor.

The results at 1 Hz extracted from Fig. 8 are shown inFig. 9 as a function of porosity. The effect on the scaffolds’stiffness of this parameter, as well as the effect of the poresize, is almost lost, as compared with the data obtainedfor the dry samples. Such behavior strengthens the hypoth-esis of the hydrostatic effect of the water within the poresthat makes difficult the deformability of the construct.There is also no apparent effect of porosity and pore sizeon the loss factor (Fig. 9b).

The mechanical properties of scaffolds are widelyreported in the literature, but most of the tests are per-formed in dry samples. In this work it is clear that the vis-coelastic properties of the studied scaffolds are differentwhen the samples are measured under physiological condi-tions. Therefore, any comparison of the mechanical perfor-mance of polymeric constructs with biomechanicalproperties of tissues should ideally made with experimental

Fig. 9. (a) Variation of E0 with porosity (d) and pore size (j) of theSPLA70 scaffolds at 1 Hz. (b) Variation of tand with porosity (d) andpore size (j) of the SPLA70 scaffolds at 1 Hz. The experiment was carriedout at 37 �C and in immersed state.

958 S. Ghosh et al. / Acta Biomaterialia 4 (2008) 950–959

data taken on immersed samples in physiological simulatedsolutions, at 37 �C. We believe that it is not enough to wetthe samples and measure them in conventional equipments;for the case of scaffolds, the samples should be completelyimmersed during the measurements.

4. Conclusions

Three-dimensional scaffolds of a blend of starch andpoly(L-lactic) acid), SPLA70, were produced by compres-sion molding followed by leaching of salt and characterizedin terms of morphology, water uptake and viscoelasticproperties using dynamic mechanical analysis. The resultsindicate that scaffolds with porosities of 74–82% can beprepared with the procedure employed. The structuresexhibited homogeneous porosities throughout the entirevolume. The hydraulic conductivity of the porous SPLA70scaffolds increases with increasing pre-seeded porogen size,as indicated with water uptake capability.

As expected, the elastic moduli of the dry scaffolds havebeen decreased with increasing porosity. However, poresize also plays an important role in the mechanical perfor-

mance of the scaffolds. Apparently, the morphology didnot influence strongly the damping properties of the scaf-folds; however, for the highest porosities the loss factorwas higher than in the other cases that exhibit values closeto human bone.

In situ DMA experiments were also performed with thesamples immersed in a physiological simulated solution of37 �C. The viscoelastic and mechanical performance of suchscaffolds measured in these conditions are significantly dif-ferent compared with dry state measurement. In immersedstate, the stiffness was much less dependent on the pore archi-tecture and porosity, probably due to the hydrostatic effectof water entrapped within the pores that could also explainthe higher values of the loss factor in the immersed samples.

From the obtained results it can be concluded thatDMA is a suitable technique to assess the solid-staterheological properties of scaffolds for tissue engineeringapplications allowing for measurements in simulatedphysiological conditions of hydration and temperature.

Acknowledgements

Financial support for this work was provided by FCT,through the POCTI and FEDER programmes (POCTI/FIS/61621/2004) and the European Union funded STREPProject HIPPOCRATES (NMP3-CT-2003-505758). S.Ghosh thanks the Portuguese Foundation for Scienceand Technology (FCT), Government of Portugal, forawarding the PhD Grant (SFRH/BD/12657/2003).

References

[1] Murphy WL, Kohn DH, Mooney DJ. Growth of continuousbonelike mineral within porous poly(lactide-co-glycolide) scaffoldsin vitro. J Biomed Mater Res 2000;50:50–8.

[2] Chupa JM, Foster AM, Sumner SR, Madihally SV, Matthew HWT.Vascular cell responses to polysaccharide materials: in vitro andin vivo evaluations. Biomaterials 2000;21:2315–22.

[3] Suzuki M, Ikada Y. Biodegradable polymers in medicine. In: ReisRL, San Roman J, editors. Biodegradable systems in tissue engineer-ing and regenerative medicine. NY: CRC Press; 2005. p. 3–12.

[4] Middleton JC, Tipton AJ. Synthetic biodegradable polymers asorthopedic devices. Biomaterials 2000;21:2335–46.

[5] Mano JF et al. Natural origin biodegradable systems in tissueengineering and regenerative medicine: present status and somemoving trends. J Roy Soc Interface 2007;4:999–1030.

[6] Athanasiou KA, Agrawal CM, Barber FA, Burkhart SS. Orthopaedicapplications for PLA-PGA biodegradable polymers. Arthroscopy1998;14:726–37.

[7] Marques AP, Cruz HR, Coutinho OP, Reis RL. Effect of starch-based biomaterials on the in vitro proliferation and viability ofosteoblast-like cells. J Mater Sci – Mater Med 2005;16:833–42.

[8] Harris LD, Kim BS, Mooney DJ. Open pore biodegradable matricesformed with gas foaming. J Biomed Mater Res 1998;42:396–402.

[9] Mikos AG, Thorsen AJ, Czerwonka LA, Bao Y, Langer R, WinslowDN, et al. Preparation and characterization of poly(L-lactic acid)foams. Polymer 1994;35:1068–77.

[10] Moroni L, De Wijn JR, Van Blitterswijk CA. 3D fiber-depositedscaffolds for tissue engineering: influence of pores geometry andarchitecture on dynamic mechanical properties. Biomaterials2006;27:974–85.

S. Ghosh et al. / Acta Biomaterialia 4 (2008) 950–959 959

[11] Mano JF, Reis RL, Cunha AM. Dynamic mechanical analysis inpolymers for medical applications. In: Reis RL, Cohn D, editors.Polymer based systems on tissue engineering, replacement andregeneration, Nato science series, Dordrecht. The Netherlands: Klu-wer; 2002. p. 139–64.

[12] Mano JF, Reis RL. Viscoelastic monitoring of starch-based bioma-terials in simulated physiological conditions. Mater Sci Eng A2004;370:321–5.

[13] Alves NM, Saiz-Arroyo C, Rodriguez-Perez MA, Reis RL, Mano JF.Microhardness of starch based biomaterials in simulated physiolog-ical conditions. Acta Biomater 2007;3:69–76.

[14] Mano JF, Koniarova D, Reis RL. Thermal properties of thermo-plastic starch/synthetic polymer blends with potential biomedicalapplicability. J Mater Sci – Mater Med 2003;14:127–35.

[15] Ghosh S, Viana JC, Reis RL, Mano JF. Effect of processingconditions on morphology and mechanical properties of injection-molded poly(L-lactic acid). Polym Eng Sci 2007;47:1141–7.

[16] Ghosh S, Viana JC, Reis RL, Mano JF. The double porogenapproach as a new technique for the fabrication of interconnected

poly(L-lactic acid) and starch based biodegradable scaffolds. J MaterSci – Mater Med 2007;18:185–93.

[17] Ghosh S, Viana JC, Reis RL, Mano JF. Bi-layered constructs basedon poly(L-lactic acid) and starch for tissue engineering of osteochon-dral defects. Mater Sci Eng C 2008;28:80–6.

[18] Hou Q, Grijpma DW, Feijen J. Porous polymeric structures for tissueengineering prepared by a coagulation, compression moulding andsalt leaching technique. Biomaterials 2003;24:1937–47.

[19] Katoh K, Tanabe T, Yamauchi K. Novel approach to fabricatekeratin sponge scaffolds with controlled pore size and porosity.Biomaterials 2004;25:4255–62.

[20] Buechner PM, Lakes RS, Swan C, Brand RA. A broadbandviscoelastic spectroscopic study of bovine bone: implications for fluidflow. Ann Biomed Eng 2001;29:719–28.

[21] Gibson LJ. Biomechanics of cellular solids. J Biomech 2005;38:377–9.[22] Blaker JJ, Maquet V, Jerome R, Boccaccini AR, Nazhat SN.

Mechanical properties of highly porous PDLLA/Bioglass� compositefoams as scaffolds for bone tissue engineering. Acta Biomater.2005;1:643–52.